Device and system for analyzing a sample, particularly blood, as well as methods of using the same

ABSTRACT

The present invention is related to the field of bio/chemical sampling, sensing, assays and applications. Particularly, the present invention is related to how to make the sampling/sensing/assay become simple to use, fast to results, highly sensitive, easy to use, using tiny sample volume (e.g., 0.5 μL or less), operated by a person without any professionals, reading by mobile-phone, or low cost, or a combination of them.

CROSS-REFERENCING

This application is a continuation of U.S. patent application Ser. No.16/653,936, filed on Oct. 15, 2019, which is a continuation of U.S.patent application Ser. No. 15/742,744, filed on Jan. 8, 2018, which isa 371 National Stage Application of PCT Application Serial No.PCT/US2016/051775, filed on Sep. 14, 2016, which claims the benefit ofU.S. Provisional Application Ser. No. 62/218,455, filed on Sep. 14,2015, 62/293,188, filed on Feb. 9, 2016, 62/305,123, filed on Mar. 8,2016, 62/369,181, filed on Jul. 31, 2016, and PCT/US2016/046437, filedon Sep. 14, 2016, which PCT claims the benefit of U.S. ProvisionalApplication Ser. No. 62/202,989, filed on Aug. 10, 2015, 62/218,455,filed on Sep. 14, 2015, 62/293,188, filed on Feb. 9, 2016, 62/305,123,filed on Mar. 8, 2016, 62/369,181, filed on Jul. 31, 2016, the contentsof which are relied upon and incorporated herein by reference in theirentirety. The entire disclosure of any publication or patent documentmentioned herein is entirely incorporated by reference.

FIELD

The present invention is related to the field of bio/chemical sampling,sensing, assays and applications.

BACKGROUND

In bio/chemical sample, particularly blood, analysis, there is a needfor the methods and devices that can accelerate the process (e.g.binding, mixing reagents, etc.) and quantify the parameters (e.g.analyte concentration, the sample volume, etc.), that can simplify thesample collection and measurement processes, that can handle sampleswith small volume, that allow an entire assay performed in less than aminute, that allow an assay performed by a smartphone (e.g. mobilephone), that allow non-professional to perform an assay her/himself, andthat allow a test result to be communicated locally, remotely, orwirelessly to different relevant parties. The present invention relatesto the methods, devices, and systems that can address these needs.

SUMMARY OF INVENTION

The following brief summary is not intended to include all features andaspects of the present invention. The present invention relates to themethods, devices, and systems that make bio/chemical sensing (including,not limited to, immunoassay, nucleic assay, electrolyte analysis, etc.)faster, more sensitive, less steps, easy to perform, smaller amount ofsamples required, less or reduced (or no) needs for professionalassistance, and/or lower cost, than many current sensing methods anddevices.

The goal of many of today's laboratory tests is to accurately determinethe absolute concentration of an analyte in a sample. For example, a redblood cell (RBC) test involves counting the number of red blood cells ina defined amount of whole blood, and then calculating the number of redblood cells per microliter of whole blood. However, such measurementscan be challenging to perform without using a specialized test center(i.e., in an “at home”, “in the pharmacy” or “point of care”environment) because such tests often require specializedinstrumentation and/or an accurate measuring device that is capable ofaccurately measuring a relatively small volume (such as an accuratepipette or the like) of a biological fluid.

Measurement of the Relevant Volume

Many assays provide the absolute concentration of an analyte in asample. However, the results of such assays become quite inaccurate whenonly a small volume (e.g., 100 nL to 10 μl, for example) is analyzed.This is because small volumes are difficult to dispense and/or measureaccurately.

In some assays, a liquid sample can be placed in between two plates thatare separated by spacers and analyzed. In theory, the volume of sampleanalyzed can be calculated by multiplying the area of the sample that isanalyzed by the thickness of the sample that is analyzed. In practice,however, such estimates are not easy to make and are quite inaccuratefor a variety of reasons. By way of example, some devices use beads tospace the plates apart, and either the beads or one of the plates isdeformable. Such devices may be prone to inaccuracy for the followingreasons:

-   -   Spherical spacers have a much smaller contact area (nearly a        point) with the plates. In such devices, because of the much        smaller contact area, for each unit of pressing force applied, a        much larger pressure is applied onto contact area of both the        plate and the spheres. This larger pressure causes the spheres        and/or the plates (if they are flexible) to deform, which        distorts any measurements.    -   Spherical spacers usually end up being randomly distributed        between two plates. Because the spherical spacers are        distributed randomly, the inter-spacer distances will vary        greatly, and some of the distances are be quite large. This        causes the spacers and/or the plates (if they are flexible) to        deform to a much greater extent in some areas relative to other,        which also distorts the results.    -   Randomly placed spacers that are close together may become        obstacles that block the movement of analytes (e.g., cells),        thereby potentially producing “clumps” of analytes or cells        which may cause even more difficulties.    -   Significant deformation of one of the plates may cause cells to        lyse, which may cause errors in cell counting efforts.    -   Volume calculations are inaccurate because the number of        spherical spacers in the area analyzed, as well as the extent to        which the spacers and/or one of the plates deforms varies from        sample to sample.    -   Deformation causes variation in the time that it takes for        molecules to diffuse to the surface of one of the plates.

In devices that uses spherical spacers, the volume of the part of thesample that has been analyzed can potentially be estimated by a)counting the spheres in the volume of the sample analyzed and b)experimentally estimate the thickness of a layer of sample (e.g., add aninternal standard, such as an immiscible liquid that contains a knownconcentration of calibrant, that can be used to calculate the distancebetween the plates). However, the extra steps are inconvenient toperform and, because the top plate and/or the spacers are significantlydeformed in use, the measurements obtained from such devices are stillnot very accurate.

In contrast, embodiments of the present method and device rely onspacers that have a substantially uniform height, a nearly uniformcross-section (e.g. a pillar with straight sidewall), and planar (i.e.,“flat”) tops, that are fixed to one or more of the plates in regularpattern in which the spacers are separated from one another by aconsistent, defined, distance (i.e., not at random positions that aregoverned by Poisson statistics). During use of some implementations ofthe present method and device, the spacers and plates are notsignificantly compressed or deformed in any dimension, at least whilethe plates are in the closed position and being pulled together bycapillary force. The present device can have many advantages in that, inuse of the present device, the volume of the part of the sample fromwhich data is obtained (i.e., the “relevant volume” or the volume of thepart of the sample in the analyzed area) can be readily calculated veryaccurately and, in some cases, can even be calculated prior toinitiating an assay, even if an unknown amount of the sample isdeposited onto the device. Because, in the closed position, the platesare substantially flat (which means that the thickness of the sample isuniform) and the number and dimensions of the spacers in the analyzedarea are known, the volume of sample in the area can be readilycalculated with high accuracy. The relevant volume sample can bedetermined without having to count the spacers in an area or estimatethe thickness of a layer of sample, after the assay has been performed.There is also need to deposit specific amount of sample into the device.Further, at the beginning of an incubation, the analyte molecules shouldbe evenly distributed throughout the relevant volume (to the extentallowed by Poisson statistics), not more concentrated in one arearelative to another.

Decreased Reaction Time

It is know that the diffusion constant of many analytes in an aqueousenvironment is very low and, as such, many assays require a lengthyincubation time (often several hours and in certain cases days),agitation and the use of agents or forces that encourage mixing. Suchassays are designed to allow an analyte to diffuse laterally from aninitial location to a remote destination on one of the plates (see,e.g., Wei et al, Nucl. Acids Res. 33: e78 and Toegl et al, J. Biomol.Tech. 2003 14: 197-204, for example). Such systems are limited becauseit may take several hours to get a result. Further, if a result isobtained, it is often difficult to say with any certainty that areaction has reached equilibrium at the time which the reaction wasterminated. This uncertainty, among other things, makes it impossible toestimate the absolute concentration of the analyte in the sample.

As will be explained in greater detail below, in some embodiments of thepresent method and device the spacer height and assay end point may bechosen to limit the amount of lateral diffusion of analytes during theassay. In these cases, such an assay (typically a binding assay) can berun in a very short time. In addition, the concentration of the analytein the sample can be estimated very accurately, even though the entiresample may not have been analyzed or may be of an unknown volume.

In these embodiments, an assay may be stopped and/or assay results maybe read at a time that is i. equal to or longer to the time that ittakes for a target entity to diffuse across the thickness of the uniformthickness layer at the closed configuration (i.e., shorter than the timethat it would take for the analyte to vertically diffuse from one plateto the other); and ii. shorter than the time that it takes the targetentity to laterally diffuse across the linear dimension of thepredetermined area of the binding site (i.e., shorter than the time thatit would take for the analyte to laterally diffuse from one side of thebinding site to other). In such “local binding” configurations, thevolume of the part of the sample from which data is obtained (the“relevant volume”) can be estimated reasonably accurately because it isthe volume of the sample that is immediately above the analyzed area.Indeed, the volume of the part of the sample from which data is obtainedmay be known before the assay is initiated. Such “local binding”embodiments have an additional advantage in that the sample and,optionally, any detection reagents are pressed into a thin layer over abinding site and, as such, binding between any analytes and/or detectionreagents should reach equilibrium more quickly than in embodiments inwhich the sample is not pressed into a thin layer, e.g., if a drop ofsample is simply placed on top of a plate with the binding site. Assuch, in many cases, binding equilibrium may be reached in a matter ofseconds rather than minutes and, as such, many assays, particularlybinding assays, can be done very quickly, e.g., in less than a minute.

Multiplexing

In addition, the “local binding” configuration allows one to performmultiplex assays without fluidically isolating the different reactionsfrom one another. In other words, multiple assays can be done in an openenvironment, without the assays being walled off from one another (i.e.,without fluidic isolation). For example, in local binding embodiments,two different analytes in the same sample can be assayed side-by-sideand, because the assay is be stopped and/or the assay results are beread prior to diffusion of the one analyte from one assay area into theother, the absolute concentrations of those analytes in the sample canbe determined separately from one another, even though they are notfluidically isolated from one another.

Being able to perform multiple assays on one sample, without fluidicisolation, by simply sandwiching a sample between two plates andperforming the assay in a diffusion-limited way has several advantages.For example, the assays can be done by simply dropping a droplet of asample (e.g., blood) of an unknown volume, spreading out the sampleacross the plates by pressing the plates together, incubating the samplefor a period of time and taking a reading from multiple sites in thedevice. In practicing this method, one does not need to transfer definedamounts of a sample into several chambers, which is difficult toimplement without an accurate fluid transfer and/or measuring device.Moreover, the assay is extremely rapid for the reasons set out above.Further, because the plates do not need to be made with “walls” themanufacture of the device is straightforward. Finally, there is norequirement for ports in any of the plates, i.e., ports that couldpotentially be used for adding or removing sample or a reagent while thedevice is in closed position.

Amplification Surface

In addition, in some embodiments of the present device and method, thedevice may contain an “amplification surface” see, e.g., a surfaceenhances the signal, e.g., fluorescence or luminescence, that isproduced by a detection agent. In some cases, the signal can enhanced bya nanoplasmonic effect (e.g., surface-enhanced Raman scattering).Examples of signal enhancement by an amplification surface aredescribed, e.g., in Li et al, Optics Express 2011 19: 3925-3936 andWO2012/024006, which are incorporated herein by reference. In somecases, the amplification surface may be a disk-coupled dots-on-pillarantenna array (D2PA), which has been described in U.S. Pat. No.9,013,690. In use, a device containing an amplification surface may asignal by 10³ fold or more, compared to a detector that is notconfigured to enhance the signal, thereby allowing analytes to bedetected with an extremely high sensitivity. In some embodiments, theamount of analyte in a relevant volume of a sample, particularlynon-cell analytes that are detected using a sandwich assay, can becounted digitally, e.g., using the methods described in WO2014144133.The use of an amplification surface, in some cases, allows the assay tobe read using a smartphone or the like.

Other Features

In embodiments of the present device, the spacers are fixed to the oneor more the plates are not able to change position or be swept away ifthe plate is immersed in an aqueous environment. The spacers are notspherical and they are not affixed to the surface of a plate via a weakforce, such as an electrostatic force, gravity or the like. In someembodiments, a plate having spacers may be a monolithic. In manyembodiments, the spacers are not pre-made and then affixed onto a plate(e.g., glued on or the like). Rather, the spacers may be grown and/oretched on a plate using an embossing and/or microfabrication (e.g., aphotolithography) process.

The parameters of the spacers (e.g., their cross-section, spacing anddensity, etc.) can be optimized so that, in the closed position, the topplate (which may be flexible) does not significantly deform over thepart of the sample that is being analyzed (the “relevant volume” of thesample). In some cases, the parameters of the spacers may be adjusteddepending on the flexibility of the top plate. For example, if the topplate is more flexible, then the spacers may be closer together.Likewise, if the top plate is less flexible, then the spacers may befurther apart.

Moreover, in use of many embodiments of the present device, analytes donot migrate directionally through the device after the device is closed.As such, in the closed configuration there may be no sorting orfractionating of the analytes, no directional, forced, flow of theanalytes through the device, (e.g., by gravity or electrophoresis), asdescribed in Austin (U.S. Pat. No. 6,632,652). In many cases there is noneed for the device to be coupled to a power supply to generate anelectromotive force. In many embodiments, there are no “obstacles” tohinder passage of an analyte (cell) while the sample is being spread,leading to analytes that are evenly distributed throughout the relevantvolume (to the extent allowed by Poisson statistics), not moreconcentrated in one area relative to another. In addition, in otherdevices, the function of the coverplate is to seal the device to preventliquid leaking out and, as such, the cover-plate is placed on top of thesubstrate plate at a time at which there is no sample on either of theplates. Such devices do not push liquid onto an open plate surface toproduce a thin layer of sample that can be analyzed. Additionally, inother devices, the key function of the pillars is to “filter” or sortnanoparticles (e.g., cells or alike). Hence the inter-pillar distance isdetermined by the nanoparticles being sorted, not for the goal of makingthe spacing between the cover plate and the substrate plate uniform.Finally, in devices such as Austin's device, the accuracy of sorting isprimarily controlled by the inter-pillar distances not the spacingbetween the plates, and controlling of the spacing between the plates isnot regarded as significant. Hence, such disclosures would not lead oneto modify plating spacing uniformity by changing pillar size, shape,inter-pillar spacing, etc.

In view of the above, the present device and method is believed toprovide an easy to use, inexpensive, easy to manufacture, and extremelyrapid way to determine the absolute concentration of an analyte (oranalytes, if the device and method are implemented in a multiplex way)in a liquid sample.

One aspect of the invention is the means that uses a pair of plates thatare movable to each other to manipulate a small volume sample or one ora plurality of reagents or both for a simpler, faster, and/or betterassaying. The manipulation includes, but limited to, reshaping a sample,forcing a sample flow, making a contact between the sample and reagent,measuring sample volume, reducing diffusion distance, increasingcollision frequency, etc. —all of them have benefit effects to certainassays. In the present invention, the special features and properties onthe plates, the special methods to handling the plates, and the specialways to handle the reagents and samples provide advantages in assaying.

One aspect of the invention is the means that make at least a portion ofa small droplet of a liquid sample deposited on a plate to become a thinfilm with a thickness that is controlled, predetermined, and uniformover large area. The uniform thickness can be as thin as less than 1 um.Furthermore, the invention allows the same uniform thickness bemaintained for a long time period without suffering evaporation toenvironment.

Another aspect of the invention is the means that utilizes thepredetermined uniform thin sample thickness formed by the invention todetermine the volume of a portion or entire of the sample without usingany pipette or alike.

Another aspect of the invention is an embodiment for the spacers (forcontrolling the spacing between two plates), that has a pillar shapewith a flat top and nearly uniform lateral cross-section. Such spacersoffers many advantages in controlling a sample thickness over thespacers of ball (beads) shape.

Another aspect of the invention is embodiments for the spacers (forcontrolling the spacing between two plates), that has a pillar shapewith a flat top and nearly uniform lateral cross-section. Such spacersoffers many advantages in controlling a sample thickness over thespacers of ball (beads) shape.

Another aspect of the invention is the means that make certain chemicalreactions (or mixing) occur predominately only in a small portion of thesample, not in the other part of the sample, without using fluidicisolation between the two portion of the sample.

Another aspect of the invention is the means that make multiple chemicalreactions (or mixing) occur predominately only in each perspective smallportion of the sample, not in the other part of the sample, withoutusing fluidic isolation between the different portion of the sample.Thus the invention allows multiplexed assaying in parallel using onesmall drop of sample without fluidic isolation between differentreaction sites.

Another aspect of the invention is the means that make assay (e.g.immunoassay, nucleic acid assay, etc.) faster. For example, a saturationincubation time (the time for the binding between molecules to reachequilibrium) is reduced from hours to less than 60 seconds.

Another aspect of the invention is the means that significantly increasethe detection sensitivity by one or a combination of several methods,which including an amplification surface, large or bright labels, etc.

Another aspect of the invention is the means that perform assaying usingvery small amount of sample, for example as small as 0.5 uL (microliter)or less.

Another aspect of the invention is the means that simplify an assay byallowing a minute body fluid deposited directly from a subject to thetesting or sample area.

Another aspect of the invention is the means that simplify and speed upan assay by pre-coating regents on plates. For example, a capture agentand a detection agent are pre-coated and dried on the plates. Anotherexample is that all required sensing reagents are pre-coated on plates,and a sensing is done by depositing a sample on the pre-coated plateswithout a need of depositing other reagents.

Another aspect of the invention is the means that make reading an assayperformed by a mobile phone.

Another aspect of the invention is the means that allow a person to testhis/her own biomarkers on their own within 60 secs by directly deposit adrop of their own body fluid (e.g. saliva) between a pair of plasticsand taking a picture with a mobile phone.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way. The drawings maynot be in scale. In the figures that present experimental data points,the lines that connect the data points are for guiding a viewing of thedata only and have no other means.

FIG. 1 is an illustration of a CROF (Compressed Regulated Open Flow)embodiment. Panel (a) illustrates a first plate and a second platewherein the first plate has spacers. Panel (b) illustrates depositing asample on the first plate (shown), or the second plate (not shown), orboth (not shown) at an open configuration. Panel (c) illustrates (i)using the two plates to spread the sample (the sample flow between theplates) and reduce the sample thickness, and (ii) using the spacers andthe plate to regulate the sample thickness at the closed configuration.The inner surface of each plate may have one or a plurality of bindingsites and or storage sites (not shown).

FIG. 2 illustrates plates with a binding site or a storage site. Panel(a) illustrates a plate having a binding site. Panel (b) illustrates aplate having a reagent storage site. Panel (c) illustrates a first platehaving a binding site and a second plate having a reagent storage site.Panel (d) illustrates a plate having multiple sites (binding sitesand/or storage site).

FIG. 3 is a flow-chart and schematic of a method for reducing assayincubation time by reducing sample thickness. Panel (a) illustrates afirst plate that has at least one binding site on a substrate surface.Panel (b) illustrates a second plate (which may have a different sizefrom the first plate). Panel (c) illustrates depositing a sample(containing target binding entity) on the substrate surface (shown) orthe cover plate (not shown), or both (not shown). Panel (d) illustratesmoving the first and second plates so that they are facing each other,and reducing the sample thickness by reducing the spacing of the innerspace between the plates. The reduced thickness sample is incubated. Thereduced sample thickness speeds up the incubation time. Some embodimentof the method uses spacers to regulate the spacing, which (spacers) arenot shown in the illustration.

FIG. 4 shows reducing binding or mixing time by reducing the samplethickness using two pates, spacers, and compression (shown incross-section). Panel (a) illustrates reducing the time for bindingentities in a sample to a binding site on a solid surface (X-(Volume toSurface)). Panel (b) illustrates reducing the time for binding entities(e.g. reagent) stored on a surface of plate to a binding site on asurface of another surface (X-(Surface to Surface)). Panel (c)illustrates reducing the time for adding reagents stored on a surface ofa plate into a sample that is sandwiched between the plate and otherplate (X-(Surface to Volume)).

FIG. 5 shows how to avoid or reduce local bending in a flexible plate.Panel (a) illustrates if the inter-spacer distance is too large for aflexible plate (the second plate, e.g. a plastic film) under a given setof sample and compress conditions, the plate has, at the closedconfiguration, a local sag (i.e. bending inward) between the twoneighboring pacers, assuming the first plate is rigid. The samplebetween the plates is not drawn. Panel (b) illustrates local bending(sag) in a flexible plate in panel (a) is reduced or virtually avoidedby using a proper inter-spacer distance and a proper compression force.The sample between the plates is not drawn.

FIG. 6 illustrates reducing effect of large dust on the plate spacing(sample thickness) regulation. Panel (a) illustrates When using tworigid plates, a dust with a thickness larger than a spacer height candestroy an intended plate spacing regulation by the spacers (hencedestroy the intended sample thickness regulation). The sample betweenthe plates is not drawn. Panel (b) illustrates using a proper flexibleplate and a proper inter-spacer distance, the effect of a dust isisolated to a small area around dust, while in other areas, the platespacing (hence the sample thickness) is regulated by the spacers not thedust. This illustration has the first plate is rigid, the second plateis flexible, and the spacers are initially fixed on the first plate.Panel (c) illustrates an illustration of using a proper flexible plateand a proper inter-spacer distance, the effect of a dust is isolated toa small area around dust, while in other areas, the plate spacing (hencethe sample thickness) is regulated by the spacers not the dust. Thisillustration has the first plate is rigid, the second plate is flexible,and the spacers are initially fixed on the second plate.

FIG. 7 illustrates reducing effects of surface flatness variation ofplate by using proper spacer arrangement and flexible plate(s). Panel(a) shows that surface flatness variation can be significantly largecompared with a desired sample thickness, causing errors in determininga sample thickness. In this illustration, only one plate has a largeflatness variation (in reality, both plates may have large flatnessvariation). The sample between the plates is not drawn. Panel (b)illustrates a surface flatness variation distance of a plate, λ, is thedistance from a local maximum to a neighboring local minimum of asurface height. Panel (c) illustrates how a small surface flatnessvariation can be achieved by making one or both plate flexible and usinga proper inter-spacer distance and proper compressing force to correct,at the closed configuration, the original surface flatness variation ofthe plate when they are at open configuration. The sample between theplates is not drawn. Panel (d) illustrates making the sample thicknessvariation less than the initial surface flatness variation of the plateby using a flexible second plate and a proper inter spacer distance. Theflexible plate follows the contour of the rigid plate. The samplebetween the plates is not drawn.

FIG. 8 illustrates plates and enclosed-spacers (well) for samplethickness regulation. Panel (a) illustrates a first plate and a secondplate, wherein the first plate has an enclosed-spacer (well). Panel (b)illustrates depositing a sample on the first plate (shown), or thesecond plate (not shown), or both (not shown) at an open configuration.Panel (c) illustrates (i) using the two plates to spread the sample (thesample flow between the plates) and reduce the sample thickness, and(ii) using the spacers and the plate to regulate the sample thickness atthe closed configuration.

FIG. 9 illustrates another embodiment that uses enclosed-spacers (well)for sample thickness regulation. Panel (a) illustrates a first plate anda second plate, wherein the first plate has an enclosed-spacer (well)and at least one spacer inside the well. Panel (b) illustratesdepositing a sample on the first plate (shown), or the second plate (notshown), or both (not shown) at an open configuration. Panel (c)illustrates (i) using the two plates to spread the sample (the sampleflow between the plates) and reduce the sample thickness, and (ii) usingthe spacers and the plate to regulate the sample thickness at the closedconfiguration. Panel (d) illustrates another embodiment of the first andsecond plates, wherein the first plate does not have a spacer inside thewell.

FIG. 10 schematically illustrates an exemplary embodiment of the presentinvention, a multiplexed detection in a single CROF device using onebinding site one plate and a plurality of storage sites on the otherplate. Panel (a) and (b) is a perspective and a cross-sectional view ofan exemplary device, respectively.

FIG. 11 schematically illustrates a further exemplary embodiment of thepresent invention, a multiplexed detection in a single CROF device usingone storage site on one plate and multiple binding sites on the otherplate. Panel (a) and (b) is a perspective and a cross-sectional view ofan exemplary device, respectively.

FIG. 12 schematically illustrates a further exemplary embodiment of thepresent invention, a multiplexed detection in a single CROF device withmultiple binding sites on one plate and multiple corresponding storagesites on another plate. Panel (a) and (b) is a perspective and across-sectional view of an exemplary device, respectively.

FIG. 13A schematically illustrate a QMAX assay that uses CROF with aspacer array of 30 um spacer height to achieve an assay with ansaturation incubation time less than 30 sec.

FIG. 13B is the measurement of signal of captured label vs incubationtime, demonstrating that the saturation incubation time of less than 30secs for a QMAX assay described in FIG. 13 a.

FIG. 14 shows experimentally measured LoD (limit of detection) for QAX &QMAX assay with 30 um gap (for CROF device) with wash (heterogeneousassay) and without wash (homogenous assay).

FIG. 15 illustrate a top view and cross-section view of (i) dropping asmall volume sample on a glass substrate, (ii) the sample area expandedat the closed configuration of CROF.

FIG. 16 illustrates the meaning of the some of the terms used herein.

FIG. 17 Spacers on a plate. Top view of photograph of (a) 46 um×46 umpillar spacer size and 54 um inter pillar distance, and (b) 10 um×70 umpillar spacer size and 10 um pillar distance; and prospect view SEM of(c) 30 um×40 um pillar spacer size of 2 um spacer height, and (d) 30um×40 um pillar spacer size of 30 um spacer height.

FIG. 18 Effects of IDS and plate thickness and materials on samplethickness. The measured sample thickness deviation and uniformity vs.inter-spacer distance (IDS) for different plate and spacer materials,different plate thickness, and different samples. The substrates of CROFdevices are non-treated 250 um thick planar PMMA (25.4 mm×25.4 mm insize. The X-Plates comprises a periodic pillar spacer array of 5 umspacer height, a rectangle shape (10×10 um pillar lateral size, nearlyuniform cross-section, and round corners), and 20 um, 50 um, 100 um, 200um, 500 um inter spacer distance, made of PMMA or PS of 25.4 mm×25.4 mmin size. Sample was 2 uL blood (dropped by direct contact with finger),saliva, or PBS (dropped by pipette), and the CROF devices were handpressed by hand pressing and rub over 1 in by 1 in area, and wereself-hold after the press. In the figure, label

is for 175 um thick PMMA using a blood sample, label

is for 175 um thick PMMA using a saliva sample, label

is for 125 um thick PS using PBS sample, label

is for 50 um thick PMMA using a blood sample, label

is for 25 um thick PS using a blood sample.

FIG. 19 Measured sample thickness deviation and uniformity vs.ISD⁴/(h^(x)E) (x=1 in the plot) value of X-Plates. ISD is inter spacingdistance, h is the height (thickness) of the material, E is the Young'smodulus of the material, x is a fitting parameter with a typical rangeof 1 to 3. In the test, the substrates of CROF devices are non-treated250 um thick PMMA (25.4 mm×25.4 mm in size), the X-Plates are 175 umthick non-treated PMMA, 125 um thick non-treated PS, 50 um thicknon-treated PMMA and 25 um thick non-treated PS (25.4 mm×25.4 mm insize), comprising a periodic pillar spacer array of 5 um spacer height,a rectangle shape (10×10 um pillar lateral size, nearly uniformcross-section, and round corners), and 20 um, 50 um, 100 um, 200 um, 500um inter spacer distance, the sample was 2 uL blood (dropped by directcontact with finger), saliva, or PBS (dropped by pipette), and the CROFdevices were hand pressed by hand pressing and rub over 1 in by 1 inarea, and were self-hold after the press. In the calculation ofISD⁴/h^(x=1)/E, Young's modulus is 2.5 GPa for PMMA, and 3.3 GPa for PS.When ISD⁴/(hE)'s value is larger than 10⁶ um³/Gpa, the performance ofCROF device become worse. In the figure, label

is for 175 um thick PMMA using a blood sample, label

is for 175 um thick PMMA using a saliva sample, label

is for 125 um thick PS using PBS sample, label

is for 50 um thick PMMA using a blood sample, label

is for 25 um thick PS using a blood sample.

FIG. 20 Measured sample thickness deviation and uniformity vs. interspacer distance for different pillar spacer's size and height on theX-Plates. The substrate plates of CROF devices are non-treated 1 mmthick Glass (25.4 mm×25.4 mm in size), the X-Plates are 125 um thicknon-treated PS (25.4 mm×25.4 mm in size), comprising a periodic pillarspacer array of 5 um spacer height with a rectangle shape of 10×10 umpillar lateral size (nearly uniform cross-section, and round corners)with 20 um, 50 um, 100 um, 200 um, 500 um inter spacer distance (label

), 40×40 um pillar lateral size with 60 um, 150 um and 200 um interspacer distance (label

); a periodic pillar spacer array of 12 um spacer height with arectangle shape of 40×40 urn pillar lateral size with 150 um and 200 uminter spacer distance (label

), a periodic pillar spacer array of 22 um spacer height with arectangle shape of 40×40 um pillar lateral size with 150 um and 200 uminter spacer distance (label

); the sample was 2 uL for 5 um thick CROF, 5 uL for 12 um thick CROFand 9 uL for 22 um thick CROF PBS (dropped by pipette), and the CROFdevices were hand pressed by hand pressing and rub over 1 in by 1 inarea, and were self-hold after the press. (Lines in figures are foreye-guiding purpose.)

FIG. 21 Measured sample thickness deviation and uniformity vs. differentratio of pillar width to pillar height while keep ISD for all thesamples less than 150 um. The substrates of CROF devices are non-treated1 mm thick Glass (25.4 mm×25.4 mm in size). The CROF devices were handpressed by hand pressing and rub over 1 in by 1 in area, and wereself-hold after the press. Sample in the above figures with label asfollowing:

A. X-Plate made of PS with 125 um thick (with label

), from left to right: 1: X-Plate pillar size 10×10 um, height 22 um,ISD 100 um, 9 uL PBS buffer, Ratio (w/h)=0.45; 2: X-Plate pillar size10×10 um, height 12 um, ISD 100 um, 5 uL PBS buffer, Ratio (w/h)=0.83;3: X-Plate pillar size 40×40 um, height 22 um, ISD 150 um, 9 uL PBSbuffer, Ratio (w/h)=1.81; 4: X-Plate pillar size 40×40 um, height 5 um,ISD 100 um, 2 uL PBS buffer, Ratio (w/h)=2; 5: X-Plate pillar size 40×40um, height 12 um, ISD 150 um, 5 uL PBS buffer, Ratio (w/h)=3.33; 6:X-Plate pillar size 40×40 urn, height 5 um, ISD 150 um, 2 uL PBS buffer,Ratio (w/h)=8; 7: X-Plate pillar size 70×70 um, height 5 um, ISD 150 um,2 uL PBS buffer, Ratio (w/h)=14

B. X-Plate made of PMMA with 175 um thick (with label

), from left to right: 1: X-Plate pillar size 10×10 um, height 22 um,ISD 100 um, 5 uL blood, Ratio (w/h)=0.45; 2: X-Plate pillar size 10×10um, height 5 um, ISD 50 um, 2 uL blood, Ratio (w/h)=2; 3: X-Plate pillarsize 30×30 um, height 30 um, ISD 80 um, 12 uL blood, Ratio (w/h)=1; 4:X-Plate pillar size 30×30 um, height 10 um, ISD 80 um, 1 uL blood, Ratio(w/h)=3; 5: X-Plate pillar size 30×30 um, height 2 um, ISD 80 um, 1 uLblood, Ratio (w/h)=15.

C. X-Plate made of PMMA with 50 um thick (with label

), from left to right: 1: X-Plate pillar size 10×10 um, height 5 um, ISD50 um, 2 uL blood, Ratio (w/h)=2.

D. X-Plate made of PS with 25 um thick (with label

), from left to right: 1: X-Plate pillar size 10×10 um, height 5 um, ISD50 um, 2 uL blood, Ratio (w/h)=2.

FIG. 22 Measured sample thickness deviation and uniformity vs. interspacer distance and pillar size/height of X-Plates, with the substratesof CROF devices are non-treated 1 mm thick Glass (25.4 mm×25.4 mm insize), the X-Plates are 125 um thick non-treated PS (25.4 mm×25.4 mm insize), comprising a periodic pillar spacer array of 5 um spacer heightwith a rectangle shape of 10×10 um pillar lateral size (nearly uniformcross-section, and round corners) with 20 um, 50 um, 100 um, 200 um, 500um inter spacer distance (label

), 40×40 um pillar lateral size with 60 um, 150 um and 200 um interspacer distance (label

); a periodic pillar spacer array of 12 um spacer height with arectangle shape of 40×40 um pillar lateral size with 60 um, 150 um and200 um inter spacer distance (label

); a periodic pillar spacer array of 22 um spacer height with arectangle shape of 40×40 um pillar lateral size with 150 um and 200 uminter spacer distance (label

); the sample was 2 uL for 5 um thick CROF, 5 uL for 12 um thick CROFand 9 uL for 22 um thick CROF PBS (dropped by pipette), and the CROFdevices were hand pressed by hand pressing and rub over 1 in by 1 inarea, and were self-hold after the press. (Lines in figures are foreye-guiding purpose.)

FIG. 23 Measured sample thickness deviation and uniformity vs. differentX-Plate thickness (25 um to 525 um) but fixed pillar size (30×38 um),pillar height (2 um) and inter spacing distances (80×82 um) made ofnon-treated PMMA, where the substrate is a 1 mm thick non-treated Glass(25.4 mm×25.4 mm in size), the sample was 1 uL blood dropped by directcontact with finger, and the CROF devices were hand pressed by handpressing and rub over 1 in by 1 in area, and were self-hold after thepress.

FIG. 24 shows measured spacing size deviation/uniformity of CROF device(different combination pairs of hydrophilic-hydrophilic with label

, hydrophilic-hydrophobic with label

) with blood volume from 0.1 uL to 0.5 uL, but same X-Plate pillar size(30×38 um), pillar height (2 um) and inter spacing distances (80×82 um),where the substrate is a 1 mm thick Glass (25.4 mm×25.4 mm in size) andthe X-Plate is made of 175 um thick PMMA (25.4 mm×25.4 mm in size. Theblood was dropped by direct contact with finger, and the CROF deviceswere hand pressed by hand pressing and rub over 1 in by 1 in area.

FIG. 25 Measured sample thickness deviation and uniformity vs.substrates of non-treated 1 mm thick Glass with label

or non-treated 250 um thick PMMA with label

(25.4 mm×25.4 mm in size), where the X-Plate is a 175 um thicknon-treated PMMA (25.4 mm×25.4 mm in size) comprising a periodic pillarspacer array of 5 um spacer height, a rectangle shape (10×10 um pillarlateral size, nearly uniform cross-section, and round corners), and 50um, 100 um, 200 um and 500 um inter spacer distance, the sample was 2 uLblood dropped by direct contact with finger, and the CROF devices werehand pressed by hand pressing and rub over 1 in by 1 in area, and wereself-hold after the press.

FIG. 26 Measured sample thickness deviation and uniformity vs. tests atdifferent hand pressing time of 0 s to 60 s, where the substrate of CROFdevices is non-treated 250 um thick PMMA (25.4 mm×25.4 mm in size), theX-Plate is a 175 um thick non-treated PMMA (25.4 mm×25.4 mm in size)comprising a periodic pillar spacer array of 2 um spacer height, arectangle shape (30×38 um pillar lateral size, nearly uniformcross-section, and round corners), and 80 um inter spacer distance, thesample was 1 uL blood deposited by direct contact, and the CROF deviceswere hand pressed by hand pressing and rub over 1 in by 1 in area, andwere self-hold after the press.

FIG. 27 Measured sample thickness deviation and uniformity vs. theaverage IDS for using random ball spacer or regular pillar spacer(X-Plate), where the substrate of CROF devices is non-treated 1 mm thickGlass (25.4 mm×25.4 mm in size), the X-Plate is a 175 um thicknon-treated PMMA (25.4 mm×25.4 mm in size) comprising a periodic pillarspacer array of 5 um spacer height, a rectangle shape (10×10 um pillarlateral size, nearly uniform cross-section, and round corners), and 20um, 50 um and 100 um inter spacer distance, the sample was 2 uL PBS, andthe CROF devices were hand pressed by hand pressing and rub over 1 in by1 in area, and were self-hold after the press. The ball is soda limemicrospheres with average diameter of 4 um (5% size variation) in PBS.The microspheres are distributed in PBS with concentrations of 4×10⁵/uL,0.9×10⁵/uL, and 0.2×10⁵/uL, which corresponding to 20 um, 50 um and 100um average inter spacer distance after press. Two kinds of cover plateare used, non-treated 220 um thick Glass (25.4 mm×25.4 mm in size) andnon-treated 175 um thick PMMA (25.4 mm×25.4 mm in size). The all deviceswere pressed by hand pressing and rub over 1 in by 1 in area, and wereself-hold after the press. Label

is for using X-Plate, label

is for using beads as spacer and 220 um thick Glass slide as coverplate, label

is for using beads as spacer and 175 um thick PMMA film as cover plate.

FIG. 28 Measured sample thickness deviation and uniformity vs. differentX-Plate thickness (25 um to 350 um) and substrate thickness (25 um to750 um). X-Plates have fixed pillar size (30×38 um), pillar height (10um) and inter spacing distances (80×82 um) made of non-treated PMMA withthickness of 25 um, 175 um and 350 um, where the substrate is made ofnon-treated PMMA (25.4 mm×25.4 mm in size) with thickness of 25 um, 50um, 175 um, 250 um and 750 um. The sample was 4 uL blood dropped bydirect contact with finger, and the CROF devices were hand pressed byhand pressing and rub over 1 in by 1 in area, and were self-hold afterthe press. In the figure, label

is for using 25 um thick X-Plate, label

is for using 175 um thick X-Plate, label

is for 350 um thick X-Plate.

FIG. 29 shows (a) the microscope photo (40×) of blood cells in X-deviceswith plate spacing (hence a sample thickness) of 1 um, 2 um, 3 um and 5um. 1 um spacing X-device lyses most (99%) of the RBCs, remainsplatelets unlysed. 2 um spacing X-device separates each RBC well andmakes RBCs single layer. Some stacked RBCs are observed in 3 um spacingX-device, and much more stacked RBCs in 5 um spacing X-device. Singlelayer cell (2 um X-device) is preferred for counting. And (b) the ratioof the red blood cell area (measured from 2D top view image) to thetotal lateral area of CROF plate. The maximum at 2 um plate spacing(i.e. sample thickness), because below 2 um some RBC are lysed andhigher than 2 um the RBCs are overlapped and rotated, all of them givessmaller RBC area in the 2D image.

FIG. 30 Schematic of the BCI (Blood-cell-counting by CROF and Imaging)by smartphone (a) and photographs of the device (b). In a blood testusing the smartphone-BCI, one person first has a card (1) and pricksher/finger (2), then deposits a small amount of blood directly from thefinger onto the CROF-Card by touching the card (2), closes the card (3)and presses by a finger (4) and release it (5), inserts the card intothe optical adapter (5), and finally takes a picture of the card usingthe smartphone (6), and from the pictures taken, the software measuresthe blood volume and the blood cell counts and other parameters (6). (b)Photo of an actual smartphone and the adapter for the p-BCI.

FIG. 31 Bright-field optical microscopy images of fresh (a) and stored(b) undiluted whole blood in the CROF-Card with different final gaps,and illustration of RBCs behavior for different confinement gap. Thefresh blood has anticoagulant and was taken from a pricked finger andthe stored blood has anticoagulant and was from a commercial vendor.(a-1 to a-6) and (b-1 to b-6): for g=2, 2.2, 2.6, 3, 5, and 10 um,respectively. (c) shows cross-sectional and top-view schematics of (1)RBCs are separated from others, have no observable overlap in CROF with2 um gap, while (2) RBCs overlap each other in CROF with gap larger than2 um.

FIG. 32 Bright-field (1) and fluorescence (2) images of the same sample(fresh blood in CROF-Card taken) by smartphone with optical adapter (a)and by a high resolution microscope with DSLR camera (b). The imagesshow that the smartphone with the optical adopter has similar bloodcells photo quality as that of the high-resolution microscope andcamera.

FIG. 33 shows the measured optical intensity of one typical WBC and PLTvs their locations of these separated cells. WBC has a diameter (FWHM)around 12 um, while PLT has a diameter (FWHM) around 2 um. The maximumintensity of WBC is around 3 times larger than PLT. Both the intensityand area give WBC's overall intensity around 108 times larger thanPLT's. Thus, if using lower magnification (as 4×), WBC's area becomesmaller and its overall intensity become lower. PLT's signal will benegligible in that case.

FIG. 34 shows (a) a scatter plot of intensity of the green channel lightvs that of the red channel intensities; and (b) histogram of red/greenchannel intensity ratios for 594 WBCs within the images. From this imagewe can clearly see that the cells cluster into three distinct regions(shaded areas provided as guides for the eye), corresponding to thethree main white cell subpopulations.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The following detailed description illustrates some embodiments of theinvention by way of example and not by way of limitation. The sectionheadings and any subtitles used herein are for organizational purposesonly and are not to be construed as limiting the subject matterdescribed in any way. The contents under a section heading and/orsubtitle are not limited to the section heading and/or subtitle, butapply to the entire description of the present invention.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentclaims are not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided can be differentfrom the actual publication dates which can need to be independentlyconfirmed.

The present invention is related to, among other things, methods,devices, and systems that can improve and/or speed up thequantification, binding, and/or sensing of an analyte and/or entity in asample.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, some exemplarymethods and materials are now described.

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”,“nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence” and“oligonucleotide” are used interchangeably, and can also include pluralsof each respectively depending on the context in which the terms areutilized.

The term “capture agent” as used herein, refers to a binding member,e.g. nucleic acid molecule, polypeptide molecule, or any other moleculeor compound, that can specifically bind to its binding partner, e.g., asecond nucleic acid molecule containing nucleotide sequencescomplementary to a first nucleic acid molecule, an antibody thatspecifically recognizes an antigen, an antigen specifically recognizedby an antibody, a nucleic acid aptamer that can specifically bind to atarget molecule, etc.

The term “a secondary capture agent” which can also be referred to as a“detection agent” refers a group of biomolecules or chemical compoundsthat have highly specific affinity to the antigen. The secondary captureagent can be strongly linked to an optical detectable label, e.g.,enzyme, fluorescence label, or can itself be detected by anotherdetection agent that is linked to an optical detectable label throughbioconjugation (Hermanson, “Bioconjugate Techniques” Academic Press, 2ndEd., 2008).

The term “capture agent-reactive group” refers to a moiety of chemicalfunction in a molecule that is reactive with capture agents, i.e., canreact with a moiety (e.g., a hydroxyl, sulfhydryl, carboxyl or aminegroup) in a capture agent to produce a stable strong, e.g., covalentbond.

The terms “specific binding” and “selective binding” refer to theability of a capture agent to preferentially bind to a particular targetanalyte that is present in a heterogeneous mixture of different targetanalytes. A specific or selective binding interaction will discriminatebetween desirable (e.g., active) and undesirable (e.g., inactive) targetanalytes in a sample, typically more than about 10 to 100-fold or more(e.g., more than about 1000- or 10,000-fold).

The term “sample” as used herein relates to a material or mixture ofmaterials containing one or more analytes or entity of interest. Inparticular embodiments, the sample may be obtained from a biologicalsample such as cells, tissues, bodily fluids, and stool. Bodily fluidsof interest include but are not limited to, amniotic fluid, aqueoushumour, vitreous humour, blood (e.g., whole blood, fractionated blood,plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), cerumen(earwax), chyle, chime, endolymph, perilymph, feces, gastric acid,gastric juice, lymph, mucus (including nasal drainage and phlegm),pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva,sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomit,urine and exhaled condensate. In particular embodiments, a sample may beobtained from a subject, e.g., a human, and it may be processed prior touse in the subject assay. For example, prior to analysis, theprotein/nucleic acid may be extracted from a tissue sample prior to use,methods for which are known. In particular embodiments, the sample maybe a clinical sample, e.g., a sample collected from a patient.

The term “analyte” refers to a molecule (e.g., a protein, peptides, DNA,RNA, nucleic acid, or other molecule), cells, tissues, viruses, andnanoparticles with different shapes.

The term “assaying” refers to testing a sample to detect the presenceand/or abundance of an analyte.

As used herein, the terms “determining,” “measuring,” and “assessing,”and “assaying” are used interchangeably and include both quantitativeand qualitative determinations.

As used herein, the term “light-emitting label” refers to a label thatcan emit light when under an external excitation. This can beluminescence. Fluorescent labels (which include dye molecules or quantumdots), and luminescent labels (e.g., electro- or chemi-luminescentlabels) are types of light-emitting label. The external excitation islight (photons) for fluorescence, electrical current forelectroluminescence and chemical reaction for chemi-luminescence. Anexternal excitation can be a combination of the above.

The phrase “labeled analyte” refers to an analyte that is detectablylabeled with a light emitting label such that the analyte can bedetected by assessing the presence of the label. A labeled analyte maybe labeled directly (i.e., the analyte itself may be directly conjugatedto a label, e.g., via a strong bond, e.g., a covalent or non-covalentbond), or a labeled analyte may be labeled indirectly (i.e., the analyteis bound by a secondary capture agent that is directly labeled).

The terms “hybridizing” and “binding”, with respect to nucleic acids,are used interchangeably.

The term “capture agent/analyte complex” is a complex that results fromthe specific binding of a capture agent with an analyte. A capture agentand an analyte for the capture agent will usually specifically bind toeach other under “specific binding conditions” or “conditions suitablefor specific binding”, where such conditions are those conditions (interms of salt concentration, pH, detergent, protein concentration,temperature, etc.) which allow for binding to occur between captureagents and analytes to bind in solution. Such conditions, particularlywith respect to antibodies and their antigens and nucleic acidhybridization are well known in the art (see, e.g., Harlow and Lane(Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1989) and Ausubel, et al, Short Protocols inMolecular Biology, 5th ed., Wiley & Sons, 2002).

A subject may be any human or non-human animal. A subject may be aperson performing the instant method, a patient, a customer in a testingcenter, etc.

An “analyte,” as used herein is any substance that is suitable fortesting in the present method.

As used herein, a “diagnostic sample” refers to any biological samplethat is a bodily byproduct, such as bodily fluids, that has been derivedfrom a subject. The diagnostic sample may be obtained directly from thesubject in the form of liquid, or may be derived from the subject byfirst placing the bodily byproduct in a solution, such as a buffer.Exemplary diagnostic samples include, but are not limited to, saliva,serum, blood, sputum, urine, sweat, lacrima, semen, feces, breath,biopsies, mucus, etc.

As used herein, an “environmental sample” refers to any sample that isobtained from the environment. An environmental sample may includeliquid samples from a river, lake, pond, ocean, glaciers, icebergs,rain, snow, sewage, reservoirs, tap water, drinking water, etc.; solidsamples from soil, compost, sand, rocks, concrete, wood, brick, sewage,etc.; and gaseous samples from the air, underwater heat vents,industrial exhaust, vehicular exhaust, etc. Typically, samples that arenot in liquid form are converted to liquid form before analyzing thesample with the present method.

As used herein, a “foodstuff sample” refers to any sample that issuitable for animal consumption, e.g., human consumption. A foodstuffsample may include raw ingredients, cooked food, plant and animalsources of food, preprocessed food as well as partially or fullyprocessed food, etc. Typically, samples that are not in liquid form areconverted to liquid form before analyzing the sample with the presentmethod.

The term “diagnostic,” as used herein, refers to the use of a method oran analyte for identifying, predicting the outcome of and/or predictingtreatment response of a disease or condition of interest. A diagnosismay include predicting the likelihood of or a predisposition to having adisease or condition, estimating the severity of a disease or condition,determining the risk of progression in a disease or condition, assessingthe clinical response to a treatment, and/or predicting the response totreatment.

A “biomarker,” as used herein, is any molecule or compound that is foundin a sample of interest and that is known to be diagnostic of orassociated with the presence of or a predisposition to a disease orcondition of interest in the subject from which the sample is derived.Biomarkers include, but are not limited to, polypeptides or a complexthereof (e.g., antigen, antibody), nucleic acids (e.g., DNA, miRNA,mRNA), drug metabolites, lipids, carbohydrates, hormones, vitamins,etc., that are known to be associated with a disease or condition ofinterest.

A “condition” as used herein with respect to diagnosing a healthcondition, refers to a physiological state of mind or body that isdistinguishable from other physiological states. A health condition maynot be diagnosed as a disease in some cases. Exemplary health conditionsof interest include, but are not limited to, nutritional health; aging;exposure to environmental toxins, pesticides, herbicides, synthetichormone analogs; pregnancy; menopause; andropause; sleep; stress;prediabetes; exercise; fatigue; chemical balance; etc. The term “biotinmoiety” refers to an affinity agent that includes biotin or a biotinanalogue such as desthiobiotin, oxybiotin, 2′-iminobiotin,diaminobiotin, biotin sulfoxide, biocytin, etc. Biotin moieties bind tostreptavidin with an affinity of at least 10-8M. A biotin affinity agentmay also include a linker, e.g., -LC-biotin, -LC-LC-Biotin, -SLC-Biotinor -PEGn-Biotin where n is 3-12.

The term “amplify” refers to an increase in the magnitude of a signal,e.g., at least a 10-fold increase, at least a 100-fold increase at leasta 1,000-fold increase, at least a 10,000-fold increase, or at least a100,000-fold increase in a signal.

The term “entity” refers to, but not limited to proteins, peptides, DNA,RNA, nucleic acid, molecules (small or large), cells, tissues, viruses,nanoparticles with different shapes, that would bind to a “bindingsite”. The entity includes the capture agent, detection agent, andblocking agent. The “entity” includes the “analyte”, and the two termsare used interchangeably.

The term “binding site” refers to a location on a solid surface that canimmobilize an entity in a sample.

The term “entity partners” refers to, but not limited to proteins,peptides, DNA, RNA, nucleic acid, molecules (small or large), cells,tissues, viruses, nanoparticles with different shapes, that are on a“binding site” and would bind to the entity. The entity, include, butnot limited to, capture agents, detection agents, secondary detectionagents, or “capture agent/analyte complex”.

The term “smart phone” or “mobile phone”, which are usedinterchangeably, refers to the type of phones that has a camera andcommunication hardware and software that can take an image using thecamera, manipulate the image taken by the camera, and communicate datato a remote place. In some embodiments, the Smart Phone has a flashlight.

The term “average linear dimension” of an area is defined as a lengththat equals to the area times 4 then divided by the perimeter of thearea. For example, the area is a rectangle, that has width w, and lengthL, then the average of the linear dimension of the rectangle is4*W*L/(2*(L+W)) (where “*” means multiply and “/” means divide). By thisdefinition, the average line dimension is, respectively, W for a squareof a width W, and d for a circle with a diameter d. The area include,but not limited to, the area of a binding site or a storage site.

The term “period” of periodic structure array refers to the distancefrom the center of a structure to the center of the nearest neighboringidentical structure.

The term “storage site” refers to a site of an area on a plate, whereinthe site contains reagents to be added into a sample, and the reagentsare capable of being dissolving into the sample that is in contract withthe reagents and diffusing in the sample.

The term “relevant” means that it is relevant to detection of analytes,quantification and/or control of analyte or entity in a sample or on aplate, or quantification or control of reagent to be added to a sampleor a plate.

The term “hydrophilic”, “wetting”, or “wet” of a surface means that thecontact angle of a sample on the surface is less than 90 degree.

The term “hydrophobic”, “non-wetting”, or “does not wet” of a surfacemeans that the contact angle of a sample on the surface is equal to orlarger than 90 degree.

The term “variation” of a quantity refers to the difference between theactual value and the desired value or the average of the quantity. Andthe term “relative variation” of a quantity refers to the ratio of thevariation to the desired value or the average of the quantity. Forexample, if the desired value of a quantity is Q and the actual value is(Q+Δ), then the Δ is the variation and the Δ/(Q+Δ) is the relativevariation. The term “relative sample thickness variation” refers to theratio of the sample thickness variation to the average sample thickness.

The term “optical transparent” refers to a material that allows atransmission of an optical signal, wherein the term “optical signal”refers to, unless specified otherwise, the optical signal that is usedto probe a property of the sample, the plate, the spacers, thescale-marks, any structures used, or any combinations of thereof.

The term “none-sample-volume” refers to, at a closed configuration of aCROF process, the volume between the plates that is occupied not by thesample but by other objects that are not the sample. The objectsinclude, but not limited to, spacers, air bubbles, dusts, or anycombinations of thereof. Often none-sample-volume(s) is mixed inside thesample.

The term “saturation incubation time” refers to the time needed for thebinding between two types of molecules (e.g. capture agents andanalytes) to reach an equilibrium. For a surface immobilization assay,the “saturation incubation time” refers the time needed for the bindingbetween the target analyte (entity) in the sample and the binding siteon plate surface reaches an equilibrium, namely, the time after whichthe average number of the target molecules (the entity) captured andimmobilized by the binding site is statistically nearly constant.

In some cases, the “analyte” and “binding entity” and “entity” areinterchangeable.

A “processor,” “communication device,” “mobile device,” refer tocomputer systems that contain basic electronic elements (including oneor more of a memory, input-output interface, central processing unit,instructions, network interface, power source, etc.) to performcomputational tasks. The computer system may be a general purposecomputer that contains instructions to perform a specific task, or maybe a special-purpose computer.

A “site” or “location” as used in describing signal or datacommunication refers to the local area in which a device or subjectresides. A site may refer to a room within a building structure, such asa hospital, or a smaller geographically defined area within a largergeographically defined area. A remote site or remote location, withreference to a first site that is remote from a second site, is a firstsite that is physically separated from the second site by distanceand/or by physical obstruction. The remote site may be a first site thatis in a separate room from the second site in a building structure, afirst site that is in a different building structure from the secondsite, a first site that is in a different city from the second site,etc.

As used herein, the term “sample collection site” refers to a locationat which a sample may be obtained from a subject. A sample collectionsite may be, for example, a retailer location (e.g., a chain store,pharmacy, supermarket, or department store), a provider office, aphysician's office, a hospital, the subject's home, a military site, anemployer site, or other site or combination of sites. As used herein,the term “sample collection site” may also refer to a proprietor orrepresentative of a business, service, or institution located at, oraffiliated with, the site.

As used herein, “raw data” includes signals and direct read-outs fromsensors, cameras, and other components and instruments which detect ormeasure properties or characteristics of a sample.

“Process management,” as used herein, refers to any number of methodsand systems for planning and/or monitoring the performance of a process,such as a sample analysis process

One with skill in the art will appreciate that the present invention isnot limited in its application to the details of construction, thearrangements of components, category selections, weightings,pre-determined signal limits, or the steps set forth in the descriptionor drawings herein. The invention is capable of other embodiments and ofbeing practiced or being carried out in many different ways.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise, e.g., when the word “single” isused. For example, reference to “an analyte” includes a single analyteand multiple analytes, reference to “a capture agent” includes a singlecapture agent and multiple capture agents, reference to “a detectionagent” includes a single detection agent and multiple detection agents,and reference to “an agent” includes a single agent and multiple agents.

Device and System for Analyzing a Sample, Particularly Blood, andMethods for Using the Same

Provided herein is a device for analyzing an analyte in a sample,particularly blood. In some embodiments, the device comprises: a firstplate and a second plate, wherein:

-   -   the plates are movable relative to each other into different        configurations (e.g., via a hinge);    -   one or both plates are flexible;    -   each of the plates has, on its respective surface, a sample        contact area for contacting a sample;    -   one or both of the plates comprise spacers that are fixed with a        respective plate, wherein the spacers have a predetermined        substantially uniform height and a predetermined constant        inter-spacer distance that is in the range of 7 um to 200 um        (e.g., 7 um to 50 um (microns), 50 um to 120 um or 120 um to 200        um) and wherein at least one of the spacers is inside the sample        contact area;

a detector that detects the analyte in the sample;

wherein one of the configurations is an open configuration, in which:the two plates are separated apart, the spacing between the plates isnot regulated by the spacers, and the sample is deposited on one or bothof the plates; and

wherein another of the configurations is a closed configuration which isconfigured after the sample deposition in the open configuration; and inthe closed configuration: at least part of the sample is compressed bythe two plates into a layer of highly uniform thickness and issubstantially stagnant (i.e., having substantially no current ordirectional flow) relative to the plates, wherein the uniform thicknessof the layer (which may have a lateral area of at least 0.1 mm², atleast 0.5 mm² or at least 1 mm²) is confined by the inner surfaces ofthe two plates and is regulated by the plates and the spacers, and hasan average thickness equal to or less than 5 um (e.g., 1.8 um to 2 um, 2um to 2.2 um, 2.2 um to 2.6 um, or 2.6 um to 3.8 um) with a smallvariation (e.g., a variation of less than 10%, less than 5% or less than1%); and wherein at the closed configuration, the detector detects theanalyte in the at least part of the sample.

As described below, the device may be used for analyzing the analytewhich comprises a molecule (e.g., a protein, peptides, DNA, RNA, nucleicacid, or other molecule), cells, tissues, viruses, and nanoparticleswith different shapes; and for example, for counting cells (e.g., redblood cells and white blood cells) in a blood sample that is placed inthe device.

In some embodiments, the device may comprise a dry reagent coated on oneor both plates. In some embodiments, the dry reagent may bind to ananalyte in the sample and immobilize the analyte on a surface on one orboth of the plates. In these embodiments, the reagent may be an antibodyor other specific binding agent, for example. This dry reagent may havea pre-determined area. In other embodiments, the device may comprise areleasable dry reagent on one or more of the plates, e.g., a labeledreagent such as a cell stain or a labeled detection agent such as anantibody or the like. In some cases, there may be a release time controlmaterial on the plate that contains the releasable dry reagent, whereinthe release time control material delays the time that the releasabledry regent is released into the sample. In some cases, the release timecontrol material delays the time that the dry regent starts is releasedinto the sample by at least 3 seconds, e.g., at least 5 seconds or atleast 10 seconds. Some embodiments, the drive may contain multiple drybinding sites and/or multiple reagent sites, thereby allowing multiplexassays to be performed. In some cases, the areas occupied by the dryingbinding sites may oppose the areas occupied by the reagent sites whenthe plates are in the closed position.

In some embodiments, the regent comprises anticoagulant and/or stainingreagent(s).

In some embodiments, the analyte may be a molecule (e.g., a protein,peptides, DNA, RNA, nucleic acid, or other molecule), cell, tissue,virus, or nanoparticles with different shapes. In some embodiments, theanalytes may be white blood cell, red blood cells and platelets. In someembodiments, the analyte is stained.

In some embodiments, the spacers regulating the layer of uniformthickness (i.e., the spacers that are spacing the plates away from eachother in the layer) have a “filling factor” of at least 1%, e.g., atleast 2% or at least 5%, wherein the filling factor is the ratio of thespacer area that is in contact with the layer of uniform thickness tothe total plate area that is in contact with the layer of uniformthickness. In some embodiments, for spacers regulating the layer ofuniform thickness, the Young's modulus of the spacers times the fillingfactor of the spacers is equal or larger than 10 MPa, e.g., at least 15MPa or at least 20 MPa, where the filling factor is the ratio of thespacer area that is in contact with the layer of uniform thickness tothe total plate area that is in contact with the layer of uniformthickness. In some embodiments, the thickness of the flexible platetimes the Young's modulus of the flexible plate is in the range 60 to750 GPa-um, e.g., 100 to 300 GPa-um, 300 to 550 GPa-um, or 550 to 750GPa-um. In some embodiments, for a flexible plate, the fourth power ofthe inter-spacer-distance (ISD) divided by the thickness of the flexibleplate (h) and the Young's modulus (E) of the flexible plate, ISD⁴/(hE),is equal to or less than 10⁶ um³/GPa, e.g., less than 10⁵ um³/GPa, lessthen 10⁴ um³/GPa or less than 10³ um³/GPa.

In some embodiments, one or both plates comprises a location markereither on a surface of or inside the plate, that provide information ofa location of the plate, e.g., a location that is going to be analyzedor a location onto which the sample should be deposited. In some cases,one or both plates may comprise a scale marker, either on a surface ofor inside the plate, that provides information of a lateral dimension ofa structure of the sample and/or the plate. In some embodiments, one orboth plates comprises an imaging marker, either on surface of or insidethe plate that assists an imaging of the sample. For example, theimaging marker could help focus the imaging device or direct the imagingdevice to a location on the device. In some embodiments, the spacers canfunction as a location marker, a scale marker, an imaging marker, or anycombination of thereof.

In some embodiments, the average thickness of the layer of uniformthickness is in the range of 2 um to 2.2 um and the sample is blood. Insome embodiments, the average thickness of the layer of uniformthickness is in the range of 2.2 um to 2.6 um and the sample is blood.In some embodiments, the average thickness of the layer of uniformthickness is in the range of 1.8 um to 2 um and the sample is blood. Insome embodiments, the average thickness of the layer of uniformthickness is in the range of 2.6 um to 3.8 um and the sample is blood.In some embodiments, the average thickness of the layer of uniformthickness is in the range of 1.8 um to 3.8 um and the sample is wholeblood without a dilution by another liquid.

In some cases, the average thickness of the layer of uniform thicknessis about equal to a minimum dimension of an analyte in the sample, e.g.,a red blood cell or another cell.

In some embodiments, the inter-spacer distance may substantiallyperiodic. In some cases, the spacers may be in a regular pattern and thespacing between adjacent spacers may be approximately the same. Thespacers may pillars with a cross-sectional shape selected from round,polygonal, circular, square, rectangular, oval, elliptical, or anycombination of the same and, in some embodiments, the spacers may have asubstantially flat top surface, wherein, for each spacer, the ratio ofthe lateral dimension of the spacer to its height is at least 1. In somecases, the minimum lateral dimension of spacer is less than orsubstantially equal to the minimum dimension of an analyte in thesample. The minimum lateral dimension of spacer is in the range of 0.5um to 100 um, e.g., in the range of 2 um to 50 um or 0.5 um to 10 um.

In some embodiments, the sample may be whole blood, e.g., blood from aclinical sample. In some cases, the blood may be obtained by drawingblood from an individual, e.g., by pricking the skin of the individual,and touching the drawn blood (without the aid of a blood transferdevice) to one of the plates. In some embodiments, the sample isundulited whole blood.

In some embodiments, the spacers have a pillar shape and the sidewallcorners of the spacers have a round shape with a radius of curvature atleast 1 um, e.g., at least 1.2 um, at least 1.5 um or at least 2.0 um.The spacers may have any convenient density, e.g., a density of at least1000/mm², e.g., a density of at least 1000/mm², a density of at least2000/mm², a density of at least 5,000/mm² or a density of at least10,000/mm².

In this device, at least one of the plates may be transparent, therebyallowing the assay to be read optically. Likewise, in this device, atleast one of the plates may be made of a flexible polymer, therebyallowing the sample to be efficiently spread by compressing the platestogether. In some embodiments, the pressure that compresses the plates,the spacers are not compressible and/or, independently, only one of theplates is flexible. The flexible plate may have a thickness in the rangeof 10 um to 200 um, e.g., 10 um to 50 um, 50 um to 150 um or 150 um to200 um. As noted above, in the closed position, the thickness of thelayer of uniform thickness may have a small variation. In someembodiments, the variation may be less than 30%, less than 20%, lessthan 10%, less than 5% or less than 2%, meaning that the thickness ofthe area does not exceed +/−30%, +/−20%, +/−10%, +/−5% or +/−2% of theaverage thickness.

In some embodiments, the first and second plates are connected and areconfigured to be changed from the open configuration to the closedconfiguration by folding the plates. In some embodiments, the first andsecond plates can be connected by a hinge and are configured to bechanged from the open configuration to the closed configuration byfolding the plates such that the device bends along the hinge. The hingemay be a separate material that is attached to the plates or, in somecases, the plates may be integral with the plates. In some cases, thefirst and second plates are made in a single piece of material and areconfigured to be changed from the open configuration to the closedconfiguration by folding the plates, e.g., along a hinge.

In some embodiments, the device is configured to analyze the sample veryrapidly. In some cases, the analysis may be done in 60 seconds or less,in 30 seconds, in 20 seconds or less or in 10 seconds or less.

In any embodiments, the dry binding site may comprise a capture agentsuch as an antibody or nucleic acid. In some embodiments, the releasabledry reagent may be a labeled reagent such as a fluorescently-labeledreagent, e.g., a fluorescently-labeled antibody or a cell stain suchRomanowsky's stain, Leishman stain, May-Grunwald stain, Giemsa stain,Jenner's stain, Wright's stain, or any combination of the same (e.g.,Wright-Giemsa stain). Such a stain may comprise eosin Y or eosin B withmethylene blue. In certain embodiments, the stain may be an alkalinestain such as haematoxylin.

In some embodiments, the detector may be is an optical detector thatdetects an optical signal. In some embodiments, the detector may be anelectric detector that detects an electrical signal

In some embodiments, the spacing is fixed on a plate by directlyembossing the plate or injection molding of the plate.

In some embodiments, the plate and the spacers are composted ofpolystyrene, PMMA, PC, COC, COP, or another plastic.

Also provided is a system for rapidly analyzing a sample using a mobilephone. In certain embodiments, this system may comprise: (a) a device asdescribed above; (b) a mobile communication device (e.g., a mobile phonesuch as an iphone or the like) comprising: i. one or a plurality ofcameras for the detecting and/or imaging the sample; ii. electronics,signal processors, hardware and software for receiving and/or processingthe detected signal and/or the image of the sample and for remotecommunication; and (c) a light source from either the mobilecommunication device or an external source. In some cases, the detectorin the device may be provided by the mobile communication device, anddetects an analyte in the sample at the closed configuration.

In this system, one of the plates may have a binding site that binds ananalyte, wherein at least part of the uniform sample thickness layer isover the binding site, and is substantially less than the averagelateral linear dimension of the binding site.

In some embodiments, the system may additionally comprise (d) a housingconfigured to hold the sample and to be mounted to the mobilecommunication device. The housing may comprise optics for facilitatingthe imaging and/or signal processing of the sample by the mobilecommunication device, and a mount configured to hold the optics on themobile communication device. In some cases, an element of the optics ofthe device (e.g., a lens, filter, mirror, prism or a beamsplitter) inthe housing may be movable relative to the housing such that the samplemay be imaged in at least two channels.

In some embodiments, the mobile communication device may configured tocommunicate test results to a medical professional (e.g., an MD), amedical facility (e.g., a hospital or testing lab) or an insurancecompany. In addition, the mobile communication device may be configuredto communicate information on the subject (e.g., the subject's age,gender, weight, address, name, prior test results, prior medicalhistory, etc.) with the medical professional, medical facility orinsurance company. In certain embodiments, the mobile communicationdevice may configured to receive a prescription, diagnosis or arecommendation from a medical professional. For example, in someembodiments the mobile communication device may send assay results to aremove location where a medical professional gives a diagnosis. Thediagnosis may be communicated to the subject via the mobilecommunication device. In some embodiments, the mobile communicationdevice may be configured to communicate information of the test to acloud network, and the cloud network process the information to refinethe test results. In some embodiments, the mobile communication devicemay be configured to communicate information of the test and the subjectto a cloud network, the cloud network process the information to refinethe test results, and the refined test results will send back thesubject.

In some embodiments, the mobile communication device may containhardware and software that allows it to (a) capture an image of thesample; (b) analyze a test location and a control location in in image;and (c) compare a value obtained from analysis of the test location to athreshold value that characterizes the rapid diagnostic test. In somecases, the mobile communication device communicates with the remotelocation via a wireless or cellular network. In any embodiment, themobile communication device may be a mobile phone.

The system may be used in a method that comprises (a) depositing asample on the device of the system; (b) assaying an analyte in thesample deposited on the device to generate a result; and (c)communicating the result from the mobile communication device to alocation remote from the mobile communication device. The method maycomprise analyzing the results at the remote location to provide ananalyzed result; and communicating the analyzed result from the remotelocation to the mobile communication device. As noted above, theanalysis may be done by a medical professional at a remote location.And, in some embodiments, the mobile communication device may receive aprescription, diagnosis or a recommendation from a medical professionalat a remote location.

In this method, the analyte may be a molecule (e.g., a protein,peptides, DNA, RNA, nucleic acid, or other molecule), a cell, tissue,virus, or nanoparticle nanoparticles with different shapes, for example.In some embodiments, the analytes may be white blood cells, red bloodcells and/or platelets.

In this method, the sample may be undiluted whole blood that can betransferred directly onto the device from the site of blood draw. Insome embodiments, the blood sample is a clinical sample.

In some embodiments, the assaying step may comprise detecting an analytein the sample, e.g., a biomarker such as a protein, nucleic acid, cell,or metabolite that is in the blood. This assay may be a binding assay ora biochemical assay, for example.

In some embodiments, the method comprises counting the number of redblood cells and/or counting the number of white blood cells. In somecases, the method may comprise staining the cells in the sample andcounting the number of one or more of the neutrophils, lymphocytes,monocytes, eosinophils and basophils in the sample.

In some embodiments, the method may be used to perform a white bloodcell differential (for at least neutrophils, eosinophils andlymphocytes) in order to obtain a potential diagnosis for an infection,inflammation, allergies, asthma, an immune disorders (e.g., anautoimmune disorders or an immune deficiency), leukemia (e.g., chronicmyeloid leukemia, chronic lymphocytic leukemia), myelodysplasticsyndrome or a cyeloproliferative neoplasms (e.g., myelofibrosis).

Also provided is a method for analyzing a sample. In some embodiments,this method may comprise obtaining a device as described above,depositing the sample onto one or both pates of the device; placing theplates in a closed configuration and applying an external force over atleast part of the plates; and analyzing an analyte in the sample in thelayer of uniform thickness while the plates are the closedconfiguration.

In some embodiments, this method may comprise:

(a) obtaining a sample;

(b) obtaining a first and second plates that are movable relative toeach other into different configurations, wherein each plate has asample contact surface that is substantially planar, one or both platesare flexible, and one or both of the plates comprise spacers that arefixed with a respective sample contacting surface, and wherein thespacers have:

-   -   i. a predetermined substantially uniform height,    -   ii. a shape of pillar with substantially uniform cross-section        and a flat top surface;    -   iii. a ratio of the width to the height equal or larger than        one;    -   iv. a predetermined constant inter-spacer distance that is in        the range of 10 μm to 200 μm;    -   v. a filling factor of equal to 1% or larger; and

(c) depositing the sample on one or both of the plates when the platesare configured in an open configuration, wherein the open configurationis a configuration in which the two plates are either partially orcompletely separated apart and the spacing between the plates is notregulated by the spacers;

(d), after (c), using the two plates to compress at least part of thesample into a layer of substantially uniform thickness that is confinedby the sample contact surfaces of the plates, wherein the uniformthickness of the layer is regulated by the spacers and the plates, andhas an average value in the range of 1.8 μm to 3 μm with a variation ofless than 10%, wherein the compressing comprises:

-   -   bringing the two plates together; and    -   conformable pressing, either in parallel or sequentially, an        area of at least one of the plates to press the plates together        to a closed configuration, wherein the conformable pressing        generates a substantially uniform pressure on the plates over        the at least part of the sample, and the pressing spreads the at        least part of the sample laterally between the sample contact        surfaces of the plates, and wherein the closed configuration is        a configuration in which the spacing between the plates in the        layer of uniform thickness region is regulated by the spacers;        and

(e) analyzing the blood in the layer of uniform thickness while theplates are the closed configuration;

wherein the filling factor is the ratio of the spacer contact area tothe total plate area;

wherein a conformable pressing is a method that makes the pressureapplied over an area is substantially constant regardless the shapevariation of the outer surfaces of the plates; and

wherein the parallel pressing applies the pressures on the intended areaat the same time, and a sequential pressing applies the pressure on apart of the intended area and gradually move to other area.

In some embodiments, this method may comprise: removing the externalforce after the plates are in the closed configuration; imaging thelayer of uniform thickness while the plates are the closedconfiguration; and counting a number of analytes, e.g., cells in an areaof the image. As noted above, in these embodiments, the inter-spacerdistance may in the range of 20 um to 200 um or 5 um to 20 um. In theseembodiments, the product of the filling factor and the Young's modulusof the spacer is 2 MPa or larger. In some embodiments, the surfacevariation is less than 30 nm.

In some embodiments, the sample may be an undiluted whole blood intowhich no anticoagulant has been added. In these embodiments, thedepositing step (b) may be done by: i. pricking the skin of a humanrelease a droplet of blood onto the skin and ii. contacting the dropletof blood with one or both of the plates without use of a blood transfertool.

The analyzing step may be done by, e.g., counting the number of redblood cells and/or counting the number of white blood cells. In someembodiments, the method may comprise staining the cells in the sampleand counting the number of neutrophils, lymphocytes, monocytes,eosinophils and basophils, or any combination thereof.

In any of these embodiments, the imaging and counting may be done by: i.illuminating the cells in the layer of uniform thickness; ii. taking oneor more images of the cells using a CCD or CMOS sensor; iii. identifyingcells in the image using a computer; and iv. counting a number of cellsin an area of the image.

In some embodiments, the external force may be provided by human hand,e.g., by pressing down using a digit such as a thumb, or pinchingbetween a thumb and another digit such as a forefinger on the same hand.

In some embodiments, the method may comprise measuring sodium,potassium, chloride, bicarbonate, blood urea, nitrogen, magnesium,creatinine, glucose, calcium, HDL cholesterol LDL cholesterol levelsand/or triglyceride levels in the layer of uniform thickness. Thedetails of how to perform such assays may be adapted from known methods.

In some embodiments, one or more of the plates may comprises a dryreagent coated on one or both plates (e.g., a binding agent, a stainingagent, a detection agent or an assay reactant).

In some embodiments, the layer of uniform thickness sample may athickness uniformity of up to +/−5%, e.g., up to +/−2% or up to +/−1%.

In some embodiments, the spacers are pillars with a cross-sectionalshape selected from round, polygonal, circular, square, rectangular,oval, elliptical, or any combination of the same. In some embodiments,the spacing between the spacers is approximately the average thicknessof red blood cells.

The sample may be analyzed in a variety of different ways. For example,in some embodiments, the analyzing step comprises imaging cells, e.g.,red blood cells, while blood cells, or platelets, in the blood. Theanalysis may include imaging cancer cells, viruses, or bacterias in theblood. In some embodiments, the method may comprise analyzing the bloodcomprises detecting of proteins or nucleic acids.

In some embodiments, the analysis may comprise measuring hemocytes,which may comprise determining of the sample thickness using the spacer,determining the lateral area by imaging, and calculating the area of redblood cells using the 2D image. The method may comprise measuring redcell concentration in the blood, white blood cell concentration in theblood, and/or platelet concentration in the blood.

In any of the embodiments described above, the sample may be wholeblood.

Immunohistochemistry

In immunohistochemical (IHC) staining methods, a tissue sample is fixed(e.g., in paraformaldehyde), optionally embedding in wax, sliced intothin sections that are less then 100 um thick (e.g., 2 um to 6 umthick), and then mounted onto a support such as a glass slide. Oncemounted, the tissue sections may be dehydrated using alcohol washes ofincreasing concentrations and cleared using a detergent such as xylene.

In most IHC methods, a primary and a secondary antibody may be used. Insuch methods, the primary antibody binds to antigen of interest (e.g., abiomarker) and is unlabeled. The secondary antibody binds to the primaryantibody and directly conjugated either to a reporter molecule or to alinker molecule (e.g., biotin) that can recruit reporter molecule thatis in solution. Alternatively, the primary antibody itself may bedirectly conjugated either to a reporter molecule or to a linkermolecule (e.g., biotin) that can recruit reporter molecule that is insolution. Reporter molecules include fluorophores (e.g., FITC, TRITC,AMCA, fluorescein and rhodamine) and enzymes such as alkalinephosphatase (AP) and horseradish peroxidase (HRP), for which there are avariety of fluorogenic, chromogenic and chemiluminescent substrates suchas DAB or BCIP/NBT.

In direct methods, the tissue section is incubated with a labeledprimary antibody (e.g. an FITC-conjugated antibody) in binding buffer.The primary antibody binds directly with the antigen in the tissuesection and, after the tissue section has been washed to remove anyunbound primary antibody, the section is be analyzed by microscopy.

In indirect methods, the tissue section is incubated with an unlabeledprimary antibody that binds to the target antigen in the tissue. Afterthe tissue section is washed to remove unbound primary antibody, thetissue section is incubated with a labeled secondary antibody that bindsto the primary antibody.

After immunohistochemical staining of the antigen, the tissue sample maybe stained with another dye, e.g., hematoxylin, Hoechst stain and DAPI,to provide contrast and/or identify other features.

The present device may be used for immunohistochemical (IHC) staining atissue sample. In these embodiments, the device may comprise

a first plate and a second plate, wherein:

-   -   the plates are movable relative to each other into different        configurations;    -   one or both plates are flexible;    -   each of the plates has, on its respective surface, a sample        contact area for contacting a tissue sample or a IHC staining        liquid;    -   the sample contact area in the first plate is smooth and        planner;    -   the sample contact area in the second plate comprise spacers        that are fixed on the surface and have a predetermined        substantially uniform height and a predetermined constant        inter-spacer distance that is in the range of 7 μm to 200 μm;

wherein one of the configurations is an open configuration, in which:the two plates are completely or partially separated apart, the spacingbetween the plates is not regulated by the spacers; and

wherein another of the configurations is a closed configuration which isconfigured after a deposition of the sample and the IHC staining liquidin the open configuration; and in the closed configuration: at leastpart of the sample is between the two plates and a layer of at leastpart of staining liquid is between the at least part of the sample andthe second plate, wherein the thickness of the at least part of stainingliquid layer is regulated by the plates, the sample, and the spacers,and has an average distance between the sample surface and the secondplate surface is equal or less than 250 μm with a small variation.

In some embodiments, the device may comprise a dry IHC staining agentcoated on the sample contact area of one or both plates.

In some embodiments, the device may comprise a dry IHC staining agentcoated on the sample contact area of the second plate, and the IHCstaining liquid comprise a liquid that dissolve the dry IHC stainingagent. In some embodiments, the thickness of the sample is 2 um to 6 um.

Also provided is a system for rapidly staining and analyzing a tissuesample using a mobile phone comprising:

(a) sample, staining liquid, and device as described above, (b) a mobilecommunication device comprising:

-   -   i. one or a plurality of cameras for the detecting and/or        imaging the sample;    -   ii. electronics, signal processors, hardware and software for        receiving and/or processing the detected signal and/or the image        of the sample and for remote communication; and

(c) a light source from either the mobile communication device or anexternal source.

Also provided is a method for rapidly staining and analyzing a tissuesample using a mobile phone, comprising:

(a) depositing a tissue sample and a staining liquid on the device ofthe system described above, and placing the two plate into a closedconfiguration;

(b) obtaining a mobile phone that has hardware and software of imaging,data processing, and communication;

(c) assaying by the tissue sample deposited on the CROF device by themobile phone to generate a result; and

(c) communicating the result from the mobile phone to a location remotefrom the mobile phone.

Also provided is a method for staining a tissue sample, comprising:

(a) obtaining a tissue sample;

(b) obtaining a stain liquid;

(b) obtaining a first plate and a second plate, wherein:

-   -   the plates are movable relative to each other into different        configurations;    -   one or both plates are flexible;    -   each of the plates has, on its respective surface, a sample        contact area for contacting a tissue sample or a IHC staining        liquid;    -   the sample contact area in the first plate is smooth and        planner;    -   the sample contact area in the second plate comprise spacers        that are fixed on the surface and have a predetermined        substantially uniform height and a predetermined constant        inter-spacer distance that is in the range of 7 μm to 200 μm;

(c) depositing the tissue sample and the stain liquid on the plates whenthe plates are configured in an open configuration, wherein the openconfiguration is a configuration in which the two plates are eitherpartially or completely separated apart and the spacing between theplates is not regulated by the spacers;

(d), after (c), using the two plates to compress at least part of thetissue sample and at least part of the staining liquid into a closedconfiguration;

wherein in the closed configuration: at least part of the sample isbetween the two plates and a layer of at least part of staining liquidis between the at least part of the sample and the second plate, whereinthe thickness of the at least part of staining liquid layer is regulatedby the plates, the sample, and the spacers, and has an average distancebetween the sample surface and the second plate surface is equal or lessthan 250 μm with a small variation.

All of the benefits and advantages (e.g., an accelerated reaction,faster results, etc.) of other embodiments may be applied to thisdevice, system and method.

Further, all parameters described above in the context of otherembodiments (e.g., the size, spacing and shape of the spacers, theflexibility of the spacers and plates, and how the device and system canbe used, etc.) can be incorporated into IHC embodiments described inthis section.

For example, in some embodiments, the spacers regulating the layer ofuniform thickness (i.e., the spacers that are spacing the plates awayfrom each other in the layer) have a “filling factor” of at least 1%,e.g., at least 2% or at least 5%, wherein the filling factor is theratio of the spacer area that is in contact with the layer of uniformthickness to the total plate area that is in contact with the layer ofuniform thickness. In some embodiments, for spacers regulating the layerof uniform thickness, the Young's modulus of the spacers times thefilling factor of the spacers is equal or larger than 10 MPa, e.g., atleast 15 MPa or at least 20 MPa, where the filling factor is the ratioof the spacer area that is in contact with the layer of uniformthickness to the total plate area that is in contact with the layer ofuniform thickness. In some embodiments, the thickness of the flexibleplate times the Young's modulus of the flexible plate is in the range 60to 550 GPa-um, e.g., 100 to 300 GPa-um. In some embodiments, for aflexible plate, the fourth power of the inter-spacer-distance (ISD)divided by the thickness of the flexible plate (h) and the Young'smodulus (E) of the flexible plate, ISD⁴/(hE), is equal to or less than10⁶ um³/GPa, e.g., less than 10⁵ um³/GPa, less then 10⁴ um³/GPa or lessthan 10³ um³/GPa.

In some embodiments, one or both plates comprises a location markereither on a surface of or inside the plate, that provide information ofa location of the plate, e.g., a location that is going to be analyzedor a location onto which the section should be deposited. In some cases,one or both plates may comprise a scale marker, either on a surface ofor inside the plate, that provides information of a lateral dimension ofa structure of the section and/or the plate. In some embodiments, one orboth plates comprises an imaging marker, either on surface of or insidethe plate, that assists an imaging of the sample. For example, theimaging marker could help focus the imaging device or direct the imagingdevice to a location on the device. In some embodiments, the spacers canfunction as a location marker, a scale marker, an imaging marker, or anycombination of thereof.

In some embodiments, the inter-spacer distance may substantiallyperiodic. In some cases, the spacers may be in a regular pattern and thespacing between adjacent spacers may be approximately the same. Thespacers may pillars with a cross-sectional shape selected from round,polygonal, circular, square, rectangular, oval, elliptical, or anycombination of the same and, in some embodiments, the spacers may have asubstantially flat top surface, wherein, for each spacer, the ratio ofthe lateral dimension of the spacer to its height is at least 1. In somecases, the minimum lateral dimension of spacer is less than orsubstantially equal to the minimum dimension of an analyte in thesample. The minimum lateral dimension of spacer is in the range of 0.5um to 100 um, e.g., in the range of 2 um to 50 um or 0.5 um to 10 um.

In some embodiments, the spacers have a pillar shape and the sidewallcorners of the spacers have a round shape with a radius of curvature atleast 1 um, e.g., at least 1.2 um, at least 1.5 um or at least 2.0 um.The spacers may have any convenient density, e.g., a density of at least1000/mm², e.g., a density of at least 1000/mm², a density of at least2000/mm², a density of at least 5,000/mm² or a density of at least10,000/mm².

In this device, at least one of the plates may be transparent, therebyallowing the assay to be read optically. Likewise, in this device, atleast one of the plates may be made of a flexible polymer, therebyallowing the sample to be efficiently spread by compressing the platestogether. In some embodiments, the pressure that compresses the plates,the spacers are not compressible and/or, independently, only one of theplates is flexible. The flexible plate may have a thickness in the rangeof 20 um to 200 um, e.g., 50 um to 150 um. As noted above, in the closedposition, the thickness of the layer of uniform thickness may have asmall variation. In some embodiments, the variation may be less than10%, less than 5% or less than 2%, meaning that the thickness of thearea does not exceed +/−10%, +/−5% or +/−2% of the average thickness.

In some embodiments, the first and second plates are connected and thedevice can be changed from the open configuration to the closedconfiguration by folding the plates. In some embodiments, the first andsecond plates can be connected by a hinge and the device can be changedfrom the open configuration to the closed configuration by folding theplates such that the device bends along the hinge. The hinge may be aseparate material that is attached to the plates or, in some cases, theplates may be integral with the plates.

In some embodiments, the device may be capable analyzing the sectionvery rapidly. In some cases, the analysis may be done in 60 seconds orless, in 30 seconds, in 20 seconds or less or in 10 seconds or less.

In any embodiments, the dry binding site may comprise a capture agentsuch as an antibody or nucleic acid. In some embodiments, the releasabledry reagent may be a labeled reagent such as a fluorescently-labeledreagent, e.g., a fluorescently-labeled antibody or a cell stain suchRomanowsky's stain, Leishman stain, May-Grunwald stain, Giemsa stain,Jenner's stain, Wright's stain, or any combination of the same (e.g.,Wright-Giemsa stain). Such a stain may comprise eosin Y or eosin B withmethylene blue. In certain embodiments, the stain may be an alkalinestain such as haematoxylin.

In some embodiments, the system may additionally comprise (d) a housingconfigured to hold the sample and to be mounted to the mobilecommunication device. The housing may comprise optics for facilitatingthe imaging and/or signal processing of the sample by the mobilecommunication device, and a mount configured to hold the optics on themobile communication device. In some cases, an element of the optics ofthe device (e.g., a lens, filter, mirror, prism or a beamsplitter, maybe movable) such that the sample may be imaged in at least two channels.

In some embodiments, the mobile communication device may configured tocommunicate test results to a medical professional (e.g., an MD), amedical facility (e.g., a hospital or testing lab) or an insurancecompany. In addition, the mobile communication device may be configuredto communicate information on the subject (e.g., the subject's age,gender, weight, address, name, prior test results, prior medicalhistory, etc.) with the medical professional, medical facility orinsurance company. In certain embodiments, the mobile communicationdevice may configured to receive a prescription, diagnosis or arecommendation from a medical professional. For example, in someembodiments the mobile communication device may send assay results to aremove location where a medical professional gives a diagnosis. Thediagnosis may be communicated to the subject via the mobilecommunication device.

In some embodiments, the mobile communication device may containhardware and software that allows it to (a) capture an image of thesample; (b) analyze a test location and a control location in in image;and (c) compare a value obtained from analysis of the test location to athreshold value that characterizes the rapid diagnostic test. In somecases, the mobile communication device communicates with the remotelocation via a wireless or cellular network. In any embodiment, themobile communication device may be a mobile phone.

The system may be used in a method that comprises (a) sample on thedevice of the system; (b) assaying the sample deposited on the device togenerate a result; and (c) communicating the result from the mobilecommunication device to a location remote from the mobile communicationdevice. The method may comprise analyzing the results at the remotelocation to provide an analyzed result; and communicating the analyzedresult from the remote location to the mobile communication device. Asnoted above, the analysis may be done by a medical professional at aremote location. And, in some embodiments, the mobile communicationdevice may receive a prescription, diagnosis or a recommendation from amedical professional at a remote location.

Also provided is a method for analyzing a tissue section. In someembodiments, this method may comprise obtaining a device as describedabove, depositing the section onto one or both pates of the device;placing the plates in a closed configuration and applying an externalforce over at least part of the plates; and analyzing the sample in thelayer of uniform thickness while the plates are the closedconfiguration.

In some embodiments, this method may comprise:

(a) obtaining a tissue section;

(b) obtaining a first and second plates that are movable relative toeach other into different configurations, wherein each plate has asample contact surface that is substantially planar, one or both platesare flexible, and one or both of the plates comprise spacers that arefixed with a respective sample contacting surface, and wherein thespacers have:

i. a predetermined substantially uniform height,

-   -   ii. a shape of pillar with substantially uniform cross-section        and a flat top surface;    -   iii. a ratio of the width to the height equal or larger than        one;    -   iv. a predetermined constant inter-spacer distance that is in        the range of 10 μm to 200 μm;    -   v. a filling factor of equal to 1% or larger; and

(c) depositing the section on one or both of the plates when the platesare configured in an open configuration, wherein the open configurationis a configuration in which the two plates are either partially orcompletely separated apart and the spacing between the plates is notregulated by the spacers;

(d), after (c), using the two plates to compress at least part of thesection into a layer of substantially uniform thickness that is confinedby the sample contact surfaces of the plates, wherein the uniformthickness of the layer is regulated by the spacers and the plates, andhas an average value in the range of 1.8 μm to 3 μm with a variation ofless than 10%, wherein the compressing comprises:

-   -   bringing the two plates together; and    -   conformable pressing, either in parallel or sequentially, an        area of at least one of the plates to press the plates together        to a closed configuration, wherein the conformable pressing        generates a substantially uniform pressure on the plates over        the at least part of the sample, and the pressing spreads the at        least part of the sample laterally between the sample contact        surfaces of the plates, and wherein the closed configuration is        a configuration in which the spacing between the plates in the        layer of uniform thickness region is regulated by the spacers;        and

(e) analyzing the section in the layer of uniform thickness while theplates are the closed configuration;

wherein the filling factor is the ratio of the spacer contact area tothe total plate area;

wherein a conformable pressing is a method that makes the pressureapplied over an area is substantially constant regardless the shapevariation of the outer surfaces of the plates; and

wherein the parallel pressing applies the pressures on the intended areaat the same time, and a sequential pressing applies the pressure on apart of the intended area and gradually move to other area.

In some embodiments, this method may comprise: removing the externalforce after the plates are in the closed configuration; imaging thesection in the layer of uniform thickness while the plates are theclosed configuration. As noted above, in these embodiments, theinter-spacer distance may in the range of 20 um to 200 um or 5 um to 20um. In these embodiments, the product of the filling factor and theYoung's modulus of the spacer is 2 MPa or larger. In some embodiments,the surface variation is less than 30 nm.

In any of these embodiments, the imaging and counting may be done by: i.illuminating the section in the layer of uniform thickness; ii. takingone or more images of the section using a CCD or CMOS sensor.

In some embodiments, the external force may be provided by human hand,e.g., by pressing down using a digit such as a thumb, or pinchingbetween a thumb and another digit such as a forefinger on the same hand.

In some embodiments, one or more of the plates may comprises a dryreagent coated on one or both plates (e.g., a binding agent, a stainingagent, a detection agent or an assay reactant).

In some embodiments, the layer of uniform thickness sample may athickness uniformity of up to +/−5%, e.g., up to +/−2% or up to +/−1%.

In some embodiments, the spacers are pillars with a cross-sectionalshape selected from round, polygonal, circular, square, rectangular,oval, elliptical, or any combination of the same.

Compressed Regulated Open Flow” (CROF)

Many embodiments of the present invention manipulate the geometric size,location, contact areas, and mixing of a sample and/or a reagent using amethod, termed “compressed regulated open flow (CROF)”, and a devicethat performs CROF.

The term “compressed open flow (COF)” refers to a method that changesthe shape of a flowable sample deposited on a plate by (i) placing otherplate on top of at least a part of the sample and (ii) then compressingthe sample between two plates by pushing the two plates towards eachother; wherein the compression reduces a thickness of at least a part ofthe sample and makes the sample flow into open spaces between theplates.

The term “compressed regulated open flow” or “CROF” (or “self-calibratedcompressed open flow” or “SCOT” or “SCCOF”) refers to a particular typeof COF, wherein the final thickness of a part or entire sample after thecompression is “regulated” by spacers, wherein the spacers, that areplaced between the two plates.

The term “the final thickness of a part or entire sample is regulated byspacers” in a CROF means that during a CROF, once a specific samplethickness is reached, the relative movement of the two plates and hencethe change of sample thickness stop, wherein the specific thickness isdetermined by the spacer.

One embodiment of the method of CROF, as illustrated in FIG. 1 ,comprises:

(a) obtaining a sample, that is flowable;

(b) obtaining a first plate and a second plate that are movable relativeto each other into different configurations, wherein each plate has asample contact surface that is substantially planar, wherein one or bothof the plates comprise spacers and the spacers have a predeterminedheight, and the spacers are on a respective sample contacting surface;

(c) depositing, when the plates are configured in an open configuration,the sample on one or both of the plates; wherein the open configurationis a configuration in which the two plates are either partially orcompletely separated apart and the spacing between the plates is notregulated by the spacers; and

(d) after (c), spreading the sample by bringing the plates into a closedconfiguration, wherein, in the closed configuration: the plates arefacing each other, the spacers and a relevant volume of the sample arebetween the plates, the thickness of the relevant volume of the sampleis regulated by the plates and the spacers, wherein the relevant volumeis at least a portion of an entire volume of the sample, and whereinduring the sample spreading, the sample flows laterally between the twoplates.

The term “plate” refers to, unless being specified otherwise, the plateused in a CROF process, which a solid that has a surface that can beused, together with another plate, to compress a sample placed betweenthe two plate to reduce a thickness of the sample.

The term “the plates” or “the pair of the plates” refers to the twoplates in a CROF process.

The term “first plate” or “second plate” refers to the plate use in aCROF process.

The term “the plates are facing each other” refers to the cases where apair of plates are at least partially facing each other.

The term “spacers” or “stoppers” refers to, unless stated otherwise, themechanical objects that set, when being placed between two plates, alimit on the minimum spacing between the two plates that can be reachedwhen compressing the two plates together. Namely, in the compressing,the spacers will stop the relative movement of the two plates to preventthe plate spacing becoming less than a preset (i.e. predetermined)value. There are two types of the spacers: “open-spacers” and“enclosed-spacers”.

The term “open-spacer” means the spacer have a shape that allows aliquid to flow around the entire perimeter of the spacer and flow passthe spacer. For example, a pillar is an open spacer.

The term of “enclosed spacer” means the spacer of having a shape that aliquid cannot flow abound the entire perimeter of the spacer and cannotflow pass the spacer. For example, a ring shape spacer is an enclosedspacer for a liquid inside the ring, where the liquid inside the ringspacer remains inside the ring and cannot go to outside (outsideperimeter).

The term “a spacer has a predetermined height” and “spacers havepredetermined inter-spacer distance” means, respectively, that the valueof the spacer height and the inter spacer distance is known prior to aCROF process. It is not predetermined, if the value of the spacer heightand the inter-spacer distance is not known prior to a CROF process. Forexample, in the case that beads are sprayed on a plate as spacers, wherebeads are landed on random locations of the plate, the inter-spacerdistance is not predetermined. Another example of not predeterminedinter spacer distance is that the spacers moves during a CROF processes.

The term “a spacer is fixed on its respective plate” in a CROF processmeans that the spacer is attached to a location of a plate and theattachment to that location is maintained during a CROF (i.e. thelocation of the spacer on respective plate does not change). An exampleof “a spacer is fixed with its respective plate” is that a spacer ismonolithically made of one piece of material of the plate, and thelocation of the spacer relative to the plate surface does not changeduring CROF. An example of “a spacer is not fixed with its respectiveplate” is that a spacer is glued to a plate by an adhesive, but during ause of the plate, during CROF, the adhesive cannot hold the spacer atits original location on the plate surface and the spacer moves awayfrom its original location on the plate surface.

The term “a spacer is fixed to a plate monolithically” means the spacerand the plate behavior like a single piece of an object where, during ause, the spacer does not move or separated from its original location onthe plate.

The term “open configuration” of the two plates in a CROF process meansa configuration in which the two plates are either partially orcompletely separated apart and the spacing between the plates is notregulated by the spacers

The term “closed configuration” of the two plates in a CROF processmeans a configuration in which the plates are facing each other, thespacers and a relevant volume of the sample are between the plates, thethickness of the relevant volume of the sample is regulated by theplates and the spacers, wherein the relevant volume is at least aportion of an entire volume of the sample.

The term “a sample thickness is regulated by the plate and the spacers”in a CROF process means that for a give condition of the plates, thesample, the spacer, and the plate compressing method, the thickness ofat least a port of the sample at the closed configuration of the platescan be predetermined from the properties of the spacers and the plate.

The term “inner surface” or “sample surface” of a plate in a CROF devicerefers to the surface of the plate that touches the sample, while theother surface (that does not touch the sample) of the plate is termed“outer surface”.

The term “X-Plate” of a CROF device refers to a plate that comprisesspaces that are on the sample surface of the plate, wherein the spacershave a predetermined inter-spacer distance and spacer height, andwherein at least one of the spacers is inside the sample contact area.

The term “CROF device” refers to a device that performs a CROF process.The term “CROFed” means that a CROF process is used. For example, theterm “a sample was CROFed” means that the sample was put inside a CROFdevice, a CROF process was performed, and the sample was hold, unlessstated otherwise, at a final configuration of the CROF.

The term “CROF plates” refers to the two plates used in performing aCROF process.

The term “surface smoothness” or “surface smoothness variation” of aplanar surface refers to the average deviation of a planar surface froma perfect flat plane over a short distance that is about or smaller thana few micrometers. The surface smoothness is different from the surfaceflatness variation. A planar surface can have a good surface flatness,but poor surface smoothness.

The term “surface flatness” or “surface flatness variation” of a planarsurface refers to the average deviation of a planar surface from aperfect flat plane over a long distance that is about or larger than 10um. The surface flatness variation is different from the surfacesmoothness. A planar surface can have a good surface smoothness, butpoor surface flatness (i.e. large surface flatness variation).

The term “relative surface flatness” of a plate or a sample is the ratioof the plate surface flatness variation to the final sample thickness.

The term “final sample thickness” in a CROF process refers to, unlessspecified otherwise, the thickness of the sample at the closedconfiguration of the plates in a CORF process.

The term “compression method” in CROF refers to a method that brings twoplates from an open configuration to a closed configuration.

The term of “interested area” or “area of interest” of a plate refers tothe area of the plate that is relevant to the function that the platesperform.

The term “at most” means “equal to or less than”. For example, a spacerheight is at most 1 um, it means that the spacer height is equal to orless than 1 um.

The term “sample area” means the area of the sample in the directionapproximately parallel to the space between the plates and perpendicularto the sample thickness.

The term “sample thickness” refers to the sample dimension in thedirection normal to the surface of the plates that face each other(e.g., the direction of the spacing between the plates).

The term “plate-spacing” refers to the distance between the innersurfaces of the two plates.

The term “deviation of the final sample thickness” in a CROF means thedifference between the predetermined spacer height (determined fromfabrication of the spacer) and the average of the final samplethickness, wherein the average final sample thickness is averaged over agiven area (e.g. an average of 25 different points (4 mm apart) over 1.6cm by 1.6 cm area).

The term “uniformity of the measured final sample thickness” in a CROFprocess means the standard deviation of the measured final samplethickness over a given sample area (e.g. the standard deviation relativeto the average).

The term “relevant volume of a sample” and “relevant area of a sample”in a CROF process refers to, respectively, the volume and the area of aportion or entire volume of the sample deposited on the plates during aCROF process, that is relevant to a function to be performed by arespective method or device, wherein the function includes, but notlimited to, reduction in binding time of analyte or entity, detection ofanalytes, quantify of a volume, quantify of a concentration, mixing ofreagents, or control of a concentration (analytes, entity or reagents).

The term “some embodiments”, “in some embodiments” “in the presentinvention, in some embodiments”, “embodiment”, “one embodiment”,“another embodiment”, “certain embodiments”, “many embodiments”, oralike refers, unless specifically stated otherwise, to an embodiment(s)that is (are) applied to the entire disclosure (i.e. the entireinvention).

The term “height” or “thickness” of an object in a CROF process refersto, unless specifically stated, the dimension of the object that is inthe direction normal to a surface of the plate. For example, spacerheight is the dimension of the spacer in the direction normal to asurface of the plate, and the spacer height and the spacer thicknessmeans the same thing.

The term “area” of an object in a CROF process refers to, unlessspecifically stated, the area of the object that is parallel to asurface of the plate. For example, spacer area is the area of the spacerthat is parallel to a surface of the plate.

The term “lateral” or “laterally” in a CROF process refers to, unlessspecifically stated, the direction that is parallel to a surface of theplate.

The term “width” of a spacer in a CROF process refers to, unlessspecifically stated, a lateral dimension of the spacer.

The term “a spacer inside a sample” means that the spacer is surroundedby the sample (e.g. a pillar spacer inside a sample).

The term “critical bending span” of a plate in a CROF process refers thespan (i.e. distance) of the plate between two supports, at which thebending of the plate, for a given flexible plate, sample, andcompression force, is equal to an allowed bending. For example, if anallowed bending is 50 nm and the critical bending span is 40 um for agiven flexible plate, sample, and compression force, the bending of theplate between two neighboring spacers 40 um apart will be 50 nm, and thebending will be less than 50 nm if the two neighboring spacers is lessthan 40 um.

The term “flowable” for a sample means that when the thickness of thesample is reduced, the lateral dimension increases. For an example, astool sample is regarded flowable.

In some embodiments of the present invention, a sample under a CROFprocess do not to be flowable to benefit from the process, as long asthe sample thickness can be reduced under a CROF process. For anexample, to stain a tissue by put a dye on a surface of the CROF plate,a CROF process can reduce the tissue thickness and hence speed up thesaturation incubation time for staining by the dye.

Reducing (Shortening) Binding or Mixing Time

It is desirable to reduce the incubation/reaction time in performingassays or other chemical reactions. For example, in the surfaceimmobilization assays where a target analyte in a sample is detected bybeing captured by capture agents immobilized on a plate surface (i.e. asolid phase), it is often desirable to have a short saturationincubation time for capturing target analytes in the sample, orimmobilizing of the capture agents and detection agents in a solution ona plate surface, or both. Another example is the need to shorten thetime of coating a capture agent to a plate surface. And another exampleis the need to shorten the time of mixing a reagent into a sample.

The present invention provides the methods and devise that reduce (i.e.shorten) the saturation incubation time needed for binding an entity insample to a binding site on a solid surface (i.e. the time for an entityfrom a volume to a surface). Another aspect of the present invention isto reduce the time needed for a binding of an entity stored on a platesurface to a binding site on another plate surface (i.e. the time for anentity from one surface to another surface). Another aspect of thepresent invention is to reduce the time needed for adding/mixing of areagent stored on a surface into a volume of a sample (i.e. a time foradding/mixing a reagent from a surface into a volume of a sample).

The present invention reduces the saturation incubation time of bindingand/or mixing in an assay by using the devices and methods that spread asample (or a liquid) to a thinner thickness, thereby reducing the timefor an entity diffusing across the sample's thickness. A diffusion timeof an entity in a material (e.g. liquid or solid or semi-solid) isproportional to the square to the diffusion distance, hence a reductionof the sample thickness can reduce the diffusion distance, leading todrastically reduction of diffusion time and the saturation incubationtime. A thinner thickness (e.g. a tight confined space) also increasesthe frequency of collisions of an entity with other entities in amaterial, further enhancing a binding and a mixing. The means in thepresent invention also make the reduction of the sample's thicknessprecise, uniform, fast, simple (less operation steps) and applicable toreduce the sample thickness to micrometer or nanometer thick. Theinventions have great utilities in fast, low-cost, PoC, diagnostics andchemical/bio analysis. Several embodiments of the present invention areillustrated in FIG. 1-4 .

1.1 Reducing the Saturation Incubation Time of Binding an Entity in aSample to a Binding Site on a Solid Surface by Reducing the SampleThickness.

X1. A method for reducing the saturation incubation time of binding atarget entity in a sample to a binding site of a plate surface, asillustrated in FIGS. 1-2, 3 a, and 4 a, comprising:

-   -   (a) obtaining a sample that is flowable and contains a target        entity which is capable of diffusing in the sample;    -   (b) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        the first plate has, on its surface, a binding site that is        configured to bind the target entity, wherein one or both of the        plates comprise spacers, and each of the spacers is fixed with        its respective plate and has a predetermined height;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are either partially or completely separated apart and        the spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and a relevant        volume of the sample are between the plates, the binding site is        in contact with the relevant volume, and the thickness of the        relevant volume of the sample is regulated by the plates and the        spacers, is thinner than the maximum thickness of the sample        when the plates are in the open configuration;        -   wherein the relevant volume is a portion or an entire volume            of the sample; and        -   wherein the reduced thickness of the sample reduces the            saturation incubation time for binding of the target entity            in the relevant volume to the binding site.

For a given sample volume, a CROF reduces sample thickness but increasethe sample lateral dimension. The present invention utilize the fact toperform (a) local binding or mixing in portion of the sample, and (b)multiplexing of multiple binding or mixing sites, without a fluidicbarrier to fluidically separate a sample into different isolation liquidpockets.

X2. A device for reducing the saturation incubation time to bind targetentity in a relevant volume of a sample to a surface, as illustrated inFIGS. 1-2, 3 a, and 4 a, comprising:

-   -   a first plate and a second plate that (a) are movable relative        to each other into different configurations, (b) each plate has        a sample contact area for contacting a sample that has a target        entity in a relevant volume of the sample, (c) one of the plate        has binding site that binds the target entity, and (d) at least        one of the plates comprises spacers that have a predetermined        inter-spacer distance and height and are fixed on its respective        surface, wherein at least one of the spacers is inside the        sample contact area;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, and the spacing between the plates is not        regulated by the spacers,    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in an open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the relevant volume of the        sample are between the plates, the binding site is in contact        with the relevant volume, and the thickness of the relevant        volume of the sample is regulated by the plates and the spacers,        is thinner than the maximum thickness of the sample when the        plates are in the open configuration; wherein the relevant        volume is a portion or an entire volume of the sample; and        wherein the reduced thickness of the sample reduces the        saturation incubation time for a binding of the target entity in        the relevant volume to the binding site.

1.2 Reducing Saturation Incubation Time for a Binding of an EntityStored on One Plate Surface to a Binding Site on Another Plate Surface

X3. A method for reducing the saturation incubation time to bind anentity stored on a storage site of one plate to a relevant binding siteon another plate, as illustrated in FIGS. 1, 3 c, and 4 b, comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein a        surface of first plate has a binding site, and a surface of the        second plate has a storage site that contains an entity to be        bound to the binding site; wherein the area of the binding site        and the area of the storage site is less than that of respective        plates; and wherein one or both of the plates comprise spacers        and each of the spacers is fixed with its respective plate and        has a predetermined height;    -   (b) obtaining a transfer medium, wherein the entity on the        storage site are capable of being dissolving into the transfer        medium and diffusing in the transfer medium;    -   (c) depositing, when the plates are configured in an open        configuration, the transfer medium on one or both of the plates;        wherein the open configuration is a configuration in which the        two plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the transfer medium by bringing the        plates into a closed configuration, wherein, in the closed        configuration: the plates are facing each other, the spacers,        the binding site, the storage site and at least a portion of the        transfer medium are between the plates, the binding site and the        storage site are at least partially on top of each other, the        transfer medium contacts at least a part of the binding site and        the storage site, the thickness of the transfer medium is        regulated by the plates and the spacers, is thinner than the        maximum thickness of the transfer medium when the plates are in        the open configuration;        -   wherein the reduced thickness of the transfer medium reduces            the time for the binging of the entity stored on the second            plate to the binding site on the first plate.

X4. A device for reducing the saturation incubation time for binding anentity stored on a storage site of one plate to a binding site onanother plate, as illustrated in FIGS. 1, 3 c, and 4 b, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations, wherein a surface of        first plate has a binding site; and a surface of the second        plate has a storage site that contains an entity to be bound to        the binding site; wherein the area of the binding site and the        area of the storage site is less than that of respective plates;        and wherein one or both of the plates comprise spacers and each        of the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and a transfer medium can be deposited on one or        both of the plates, wherein the entity on the storage site are        capable of being dissolving into the transfer medium and        diffusing in the transfer medium,    -   wherein another of the configuration is a closed configuration,        which is configured after the transfer medium deposition in an        open configuration; and in the closed configuration: the plates        are facing each other, the spacers, the binding site, the        storage site and at least a portion of the transfer medium are        between the plates, the binding site and the storage site are at        least partially on top of each other, the transfer medium        contacts at least a part of the binding site and the storage        site, the thickness of the transfer medium is regulated by the        plates and the spacers, is thinner than the maximum thickness of        the transfer medium when the plates are in the open        configuration;    -   wherein the reduced thickness of the transfer medium reduces the        saturation incubation time for a binging of entity on the        storage site of the second plate to the binding site of the        first plate.

In the method of paragraph X3 and the device of paragraph X4, in someembodiments, the transfer medium comprises a liquid that allows adiffusion of the entity or a reagent or both.

In the method of paragraph X3 and the device of paragraph X4, in someembodiments, the transfer medium is a sample, where the sample containsan analyte (also termed target analyte) that binds the binding site.

In the method of paragraph X3 and the device of paragraph X4, in someembodiments, the transfer medium is a sample, where the sample containsan analyte (also termed target analyte) that binds the binding site andthe reagent is a detection agent that binds to the analytes.

1.3 Reducing the Time for Adding (Mixing) Reagent Stored on Surface intoa Liquid Sample

Many assays need to have reagents added into a sample (including aliquid). Often the concentration of the added reagents in the sample orthe liquid need to be controlled. There are needs for new methods thatare simple and/or low cost to perform such reagents addition andconcentration control. Two examples where reagents additions are neededare (a) blood cell counting where anticoagulant and/or stainingreagent(s) may be added into a blood sample, and (b) immunoassays wheredetection agents are added to bind a target analyte in solution.

One aspect of the present invention is the methods, devices, and systemsthat make the reagent addition and the reagent concentration controlsimple and/or low cost. In one embodiment of the current invention, areagent layer (e.g. dried reagent layer) is first put on a plate surfaceof a CROF device, then a sample is deposited into the CROF device, and aCROF process makes the sample in contact with the reagent and the samplethickness thinner than the thickness when the sample at the openconfiguration of the CROF plates. By reducing the sample thickness, itwould reduce the diffusion time of the reagent diffuses from the surfaceinto the entire sample, and hence it reduces the time for mixing thereagent with the sample.

X5. A method for reducing the time for mixing a reagent stored on aplate surface into a sample, as illustrated in FIGS. 1, 3 b, and 4 c,comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        the first plate has, on its surface, a storage site that        contains reagents to be added into a sample, and the reagents        are capable of being dissolving into the sample and diffusing in        the sample; and wherein one or both of the plates comprise        spacers and each of the spacers is fixed with its respective        plate and has a predetermined height;    -   (b) obtaining the sample;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers, the storage site,        and at least a portion of the sample are between the plates, the        sample contacts at least a portion of the storage site, the        thickness of the sample on the storage site is regulated by the        plates and the spacers, is thinner than the maximum thickness of        the sample when the plates are in the open configuration;    -   wherein the reduced thickness of the sample reduces the time for        mixing the reagents on the storage site with the sample.

In the method of paragraph X5, it further comprises a step of incubationwhile the plates are in the closed configuration, wherein the incubationtime is selected in such that results in a significant number of thereagents dissolved in the sample are contained in the relevant volume ofthe sample, wherein the relevant volume is the volume of the sample thatsits on the storage site and the incubation is a process to allow thereagent to dissolve and diffuse in the sample.

In the method of paragraph X5, it further comprises a step that, after(d) and while the plates are in the closed configuration, incubating fora time equal or less than a factor times the diffusion time of thereagent in the sample across the sample thickness regulated by theplates at the closed configuration, and then stopping the incubation;wherein the incubation allows the reagent to diffuse into the sample;and wherein the factor is 0.0001, 0.001, 0.01, 0.1, 1, 1.1, 1.2, 1.3,1.5, 2, 3, 4, 5, 10, 100, 1000, 10,000, or a range between any to thevalues. For example, if the factor is 1.1 and the diffusion time is 20seconds, then the incubation time is equal to or less than 22 second. Inone preferred embodiment, the factor is 0.1, 1, 1.5 or a range betweenany to the values.

X6. A device for reducing the time to add a reagent stored on a platesurface into a sample, as illustrated in FIGS. 1, 3 b, and 4 c,comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations, wherein the first        plate has, on its surface, a storage site that contains reagents        to be added into a sample, the reagents are capable of being        dissolving into the sample and diffusing in the sample; and        wherein one or both of the plates comprise spacers and each of        the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the transfer medium deposition in the        open configuration; and in the closed configuration: the plates        are facing each other, the spacers, the storage site, and at        least a portion of the sample are between the plates, the sample        contacts at least a portion of the storage site, the thickness        of the sample on the storage site is regulated by the plates and        the spacers, is thinner than the maximum thickness of the sample        when the plates are in the open configuration;    -   wherein the reduced thickness of the sample reduces the time for        mixing the reagents on the storage site with the sample.

In the method or the devices of any of paragraphs X1-6, in someembodiments, the relevant volume of the sample is the volume of thesample that sits on (i.e. on top of) the binding site or the storagesite.

In the method or the devices of any of paragraphs X1-6, in someembodiments, the relevant volume of the sample is the volume of thesample that sits on (i.e. on top of) the entire area or a partial areaof the binding site or the storage site.

In the method or the devices of any of paragraphs X1-6, in someembodiments, the ratio of the lateral dimension of the binding site orthe storage site to the sample thickness at the closed configuration is1.5 3 or larger, 3 or larger, 5 or larger, 10 or larger, 20 or larger,30 or larger, 50 or larger, 100 or larger, 200 or larger, 1000 orlarger, 10,000 or larger, or a range between any two of the values.

In the method or the devices of any of paragraphs X1-6, the ratio of thelateral dimension of the binding site or the storage site to the samplethickness at the closed configuration is between 3 and 20 in a preferredembodiment, 20 and 100 in another preferred embodiment, and 100 and 1000in another preferred embodiment, and 1000 and 10,000 in anotherpreferred embodiment.

In the method of any of paragraphs X1 and X3, in some embodiments, thefinal reduced sample thickness is significantly smaller than that of thearea of the binding site, so that the entity in the sample area that isoutside of the binding site will take longer time to bind to the bindingsite. With a proper selection of the incubation time, the entity thatbind to the binding sites will be primarily the entity in the samplevolume that sites on the binding site (i.e. the sample volume that isjust above the binding area). Then the calculation of the concentrationof the entity in the sample would be based on the sample thickness andthe binding site area.

In the method of paragraph X5, in some embodiments, the final reducedsample thickness is significantly smaller than that of the area of thestorage site, so that the entity

In the sample area that is outside of the binding site will take longertime to bind to the binding site. With a proper selection of theincubation time, the entity that bind to the binding sites will beprimarily the entity in the sample volume that sites on the binding site(i.e. the sample volume that is just above the binding area). Then thecalculation of the concentration of the entity in the sample would bebased on the sample thickness and the binding site area.

In the method of any of paragraphs X2, X4, X6, it further comprises acompressing device that bring the plates from an open configurations toa closed configurations. In some embodiments, the compressing device isone or any combination of the embodiments described in the disclosures

In the method of any of paragraphs X2, X4, X6, it further comprises acompressing device that bring the plates from an open configurations toa closed configurations, and a holding device that is configured to holdthe plates are in the closed configuration. In some embodiments, theholding device is one or any combination of the embodiments described inthe disclosures.

In the method of any of paragraphs X2, X4, X6, it further comprises acompressing device that bring the plates from an open configurations toa closed configurations, and a holding device that is configured to holdthe plates are in the closed configuration for a time of 0.001 sec orless, 0.01 sec or less, 0.1 sec or less, 1 sec or less, 5 sec or less,10 sec or less, 20 sec or less, 30 sec or less, 40 sec or less, 1 min orless, 2 min or less, 3 min or less, 5 min or less, 10 min or less, 20min or less, 30 min or less, 60 min or less, 90 min or less, 120 min orless, 180 min or less, 250 min or less, or a range between any two ofthese values.

In the method of any of paragraphs X2, X4, X6, it further comprises acompressing device that bring the plates from an open configurations toa closed configurations, and a holding device that is configured to holdthe plates are in the closed configuration for a time of, in a preferredembodiment, 0.001 sec or less, 0.01 sec or less, 0.1 sec or less, 1 secor less, 5 sec or less, 10 sec or less, 20 sec or less, 30 sec or less,40 sec or less, 1 min or less, 2 min or less, 3 min or less, or a rangebetween any two of these values.

Final Sample Thickness. The final sample thickness at the closedconfiguration of the plates may be a significant factor in reducing thesaturation incubation time. The final sample thickness after the samplethickness reduction/deformation, depending upon the properties of entityand samples as well as the applications, as discussed with respect tothe regulated spacing of the plates.

In some embodiments, The final sample thickness is less than about 0.5um (micrometer), less than about 1 um, less than about 1.5 um, less thanabout 2 um, less than about 4 um, less than about 6 um, less than about8 um, less than about 10 um, less than about 12 um, less than about 14um, less than about 16 um, less than about 18 um, less than about 20 um,less than about 25 um, less than about 30 um, less than about 35 um,less than about 40 um, less than about 45 um, less than about 50 um,less than about 55 um, less than about 60 um, less than about 70 um,less than about 80 um, less than about 90 um, less than about 100 um,less than about 110 um, less than about 120 um, less than about 140 um,less than about 160 um, less than about 180 um, less than about 200 um,less than about 250 um, less than about 300 um, less than about 350 um,less than about 400 um, less than about 450 urn, less than about 500 um,less than about 550 um, less than about 600 um, less than about 650 um,less than about 700 um, less than about 800 um, less than about 900 um,less than about 1000 um (1 mm), less than about 1.5 mm, less than about2 mm, less than about 2.5 mm, less than about 3 mm, less than about 3.5mm, less than about 4 mm, less than about 5 mm, less than about 6 mm,less than about 7 mm, less than about 8 mm, less than about 9 mm, lessthan about 10 mm, or a range between any two of the values.

In certain embodiments, the final sample thickness at the closedconfiguration is less than 0.5 um (micron), less than 1 um, less than 5um, less than 10 um, less than 20 um, less than 30 um, less than 50 um,less than 100 um, less than 200 um, less than 300 um, less than 500 um,less than 800 um, less than 200 um, less than 1 mm (millimeter), lessthan 2 mm (millimeter), less than 4 mm (millimeter), less than 8 mm(millimeter), or a range between any two of the values.

In certain embodiments, the Q-methods make the final sample thicknessuniform and flat surfaces of the first plate and the second plate areused.

In the present invention, the sample incubation are done in varioustemperatures, humidity, gas environment, and different time durations,with or without shaking.

Incubation Time. In the method of any of paragraphs X1 and X3, itfurther comprises a step that, after (d) and while the plates are in theclosed configuration, incubating for a time equal or less than a factortimes the diffusion time of the entity in the sample diffusing acrossthe sample thickness regulated by the plates at the closedconfiguration, and then stopping the incubation; wherein the incubationallows binding of the entity to the binding site; and wherein the factoris 0.0001, 0.001, 0.01, 0.1, 1, 1.1, 1.2, 1.3, 1.5, 2, 3, 4, 5, 10, 100,1000, 10,000, or a range between any to the values. For example, if thefactor is 1.1 and the diffusion time is 20 seconds, then the incubationtime is equal to or less than 22 second. In one preferred embodiment,the factor is 0.1, 1, 1.5 or a range between any to the values.

In the method of paragraphs X5, it further comprises a step that, after(d) and while the plates are in the closed configuration, incubating fora time equal or less than a factor times the diffusion time of thereagents diffusing across the sample thickness regulated by the platesat the closed configuration, and then stopping the incubation; whereinthe incubation allows binding of the entity to the binding site; andwherein the factor is 0.0001, 0.001, 0.01, 0.1, 1, 1.1, 1.2, 1.3, 1.5,2, 3, 4, 5, 10, 100, 1000, 10,000, or a range between any to the values.For example, if the factor is 1.1 and the diffusion time is 20 seconds,then the incubation time is equal to or less than 22 second. In onepreferred embodiment, the factor is 0.1, 1, 1.5 or a range between anyto the values.

The method of any of paragraphs of X1, X3 and X5, or the device of anyof paragraph of X2, X4, and X6, wherein at least one of the spacers isinside the sample contact area.

The method of any of paragraphs of X1, X3 and X5, or the device of anyof paragraph of X2, X4, and X6, wherein spacers that have apredetermined inter-spacer distance.

In the method of any of paragraphs X1, X3, X5, it further comprises astep of incubation while the plates are in the closed configuration, thesaturation incubation time is 0.001 sec or less, 0.01 sec or less, 0.1sec or less, 1 sec or less, 5 sec or less, 10 sec or less, 20 sec orless, 30 sec or less, 40 sec or less, 1 min or less, 2 min or less, 3min or less, 5 min or less, 10 min or less, 20 min or less, 30 min orless, 60 min or less, 90 min or less, 120 min or less, 180 min or less,250 min or less, or a range between any two of these values.

In the method of any of paragraphs X1, X3, X5, the saturation incubationtime at the reduced sample thickness at the closed configuration is0.001 sec or less, 0.01 sec or less, 0.1 sec or less, 1 sec or less, 5sec or less, 10 sec or less, 20 sec or less, 30 sec or less, 40 sec orless, 1 min or less, 2 min or less, 3 min or less, 5 min or less, 10 minor less, 20 min or less, 30 min or less, 60 min or less, 90 min or less,120 min or less, 180 min or less, 250 min or less, or a range betweenany two of these values.

In some embodiments, capture agents are first immobilized at the bindingsite, then the sample are in contact with the binding site and theentity in the sample are captured by the capture agents, and finallydetection agents are added to be bound with the captured entity and thea signal from the detection agents will be read (e.g. by optical methodsor electrical methods or a combination). In some embodiments, otherreagents besides of capture agents and detection agents are added (e.g.blocking agent).

In many applications such as PoC, it is desirable to have simple and/orlow-cost devices and methods to add additional reagents into a sample.One aspect of the present invention is related to simple and/or low-costdevices and methods to add additional reagents into a sample. The addedadditional reagents include detection agents, blocking agents, lightsignal enhancers, light signal quenchers, or others. In some embodimentsof the present invention, it controls the assay processes by usingdifferent release time of the reagents stored on the same location. Thedifferent release time can be attached by adding other materials thathave different dissolve rate.

In certain embodiments, the reagent concentration mixed in the samplecan be controlled by controlling the sample thickness (e.g. control theratio of the sample thickness to the storage site area and/or the mixingtime).

1. Plates, Spacers, Scale-Marks, Sample Thickness Regulation

2.1 Plate Configurations and Sample Thickness Regulation OpenConfiguration. In some embodiments, in the open configuration, the twoplates (i.e. the first plate and the second plate) are separated fromeach other. In certain embodiments, the two plates have one sideconnected together during all operations of the plates (including theopen and closed configuration), the two plates open and close similar toa book. In some embodiments, the two plates have rectangle (or square)shape and have two sides of the rectangle connected together during alloperations of the plates.

In some embodiments, the open configuration comprises a configurationthat the plates are far away from each other, so that the sample isdeposited onto one plate of the pair without a hindrance of the otherplate of the pair.

In some embodiments, the open configuration comprises a configurationthat the plates are far way, so that the sample is directly depositedonto one plate, as if the other plate does not exist.

In some embodiments, the open configuration comprises a configurationthat the pair of the plates are spaced apart by a distance at least 10nm, at least 100 nm, at least 1000 nm, at least 0.01 cm, at least 0.1cm, at least 0.5 cm, at least 1 cm, at least 2 cm, or at least 5 cm, ora range of any two of the values.

In some embodiments, the open configuration comprises a configurationthat the pair of plates are oriented in different orientations. In someembodiments, the open configuration comprises a configuration thatdefines an access gap between the pair of plates that is configured topermit sample addition.

In some embodiments, the open configuration comprises a configuration,wherein each plate has a sample contact surface and wherein at least oneof the contact surfaces of the plates is exposed when the plates are inthe one open configuration.

Closed Configuration and Sample Thickness Regulation. In presentinvention, a closed configuration of the two plates is the configurationthat a spacing (i.e. the distance) between the inner surfaces of the twoplates is regulated by the spacers between the two plates. Since theinner surfaces (also termed “sample surface”) of the plates are incontact with the sample during the compression step of a CROF process,hence at the closed configuration, the sample thickness is regulated bythe spacers.

During the process of bring the plates from an open configuration to aclosed configuration, the plates are facing each other (at least a partof the plates are facing each other) and a force is used to bring thetwo plates together. When the two plates are brought from an openconfiguration to a closed configuration, the inner surfaces of the twoplate compress the sample deposited on the plate(s) to reduce the samplethickness (while the sample has an open flow laterally between theplates), and the thickness of a relevant volume of the sample isdetermined by the spacers, the plates, and the method being used and bythe sample mechanical/fluidic property. The thickness at a closedconfiguration can be predetermined for a given sample and given spacers,plates and plate pressing method.

The term “regulation of the spacing between the inner surfaces of theplates by the spacers” or “the regulation of the sample thickness by theplates and the spacer”, or a thickness of the sample is regulated by thespacers and the plates” means that the thickness of the sample in a CROFprocess is determined by a given plates, spacers, sample, and pressingmethod.

In some embodiments, the regulated sample thickness at the closedconfiguration is the same as the height of a spacer; in this case, atthe closed configuration, the spacers directly contact both plates(wherein one plate is the one that the spacer is fixed on, and the otherplate is the plate that is brought to contact with the spacer).

In certain embodiments, the regulated sample thickness at the closedconfiguration is larger than the height of a spacer; in this case, atthe closed configuration, the spacers directly contacts only the platethat has the spacers fixed or attached on its surface, and indirectlycontact the other plate (i.e. indirect contact). The term “indirectcontact” with a plate means that the spacer and the plate is separatedby a thin sample layer, which is termed “residual sample layer” and itsthickness is termed “the residue thickness”. For given spacers andplates, a given plate pressing method, and a given sample, the residualthickness can be predetermined (predetermined means prior to reach theclosed configuration), leading to a predetermination of the samplethickness at the closed configuration. This is because the residue layerthickness is the same for the given conditions (the sample, spacers,plates, and pressing force) and can be pre-calibrated and/or calculated.The regulated sample thickness is approximately equal to the spacerheight plus the sample residue thickness.

In many embodiments, the size and shape of the pillars arepre-characterized (i.e. pre-determined) before their use. And thepre-determined information are used to for later assaying, such asdetermination of the sample volume (or relevant volume) and others.

In some embodiments, the regulating of the sample thickness includesapplying a closing (compression) force to the plates to maintain thespacing between the plates.

In some embodiments, the regulating of the sample thickness includesestablishing the spacing between the plates with the spacers, a closingforce applied to the plates, and physical properties of the sample, andoptionally wherein the physical properties of the sample include atleast one of viscosity and compressibility.

2.2 Plates

In present invention, generally, the plates of CROF are made of anymaterial that (i) is capable of being used to regulate, together withthe spacers, the thickness of a portion or entire volume of the sample,and (ii) has no significant adverse effects to a sample, an assay, or agoal that the plates intend to accomplish. However, in certainembodiments, particular materials (hence their properties) ae used forthe plate to achieve certain objectives.

In some embodiments, the two plates have the same or differentparameters for each of the following parameters: plate material, platethickness, plate shape, plate area, plate flexibility, plate surfaceproperty, and plate optical transparency.

Plate materials. The plates are made a single material, compositematerials, multiple materials, multilayer of materials, alloys, or acombination thereof. Each of the materials for the plate is an inorganicmaterial, am organic material, or a mix, wherein examples of thematerials are given in paragraphs of Mat-1 and Mat-2.Mat-1. The inorganic materials for the plates include, not limited to,glass, quartz, oxides, silicon-dioxide, silicon-nitride, hafnium oxide(HfO), aluminum oxide (AlO), semiconductors: (silicon, GaAs, GaN, etc.),metals (e.g. gold, silver, coper, aluminum, Ti, Ni, etc.), ceramics, orany combinations of thereof.Mat-2 The organic materials for the spacers include, not limited to,polymers (e.g. plastics) or amorphous organic materials. The polymermaterials for the spacers include, not limited to, acrylate polymers,vinyl polymers, olefin polymers, cellulosic polymers, noncellulosicpolymers, polyester polymers, Nylon, cyclic olefin copolymer (COC),poly(methyl methacrylate) (PMMA), polycarbonate (PC), cyclic olefinpolymer (COP), liquid crystalline polymer (LCP), polyimide (PA),polyethylene (PE), polyimide (PI), polypropylene (PP), poly(phenyleneether) (PPE), polystyrene (PS), polyoxymethylene (POM), polyether etherketone (PEEK), polyether sulfone (PES), poly(ethylene phthalate) (PET),polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidenefluoride (PVDF), polybutylene terephthalate (PBT), fluorinated ethylenepropylene (FEP), perfluoroalkoxyalkane (PFA), polydimethylsiloxane(PDMS), rubbers, or any combinations of thereof.

In some embodiments, the plates are each independently made of at leastone of glass, plastic, ceramic, and metal. In some embodiments, eachplate independently includes at least one of glass, plastic, ceramic,and metal.

In some embodiments, one plate is different from the other plate inlateral area, thickness, shape, materials, or surface treatment. In someembodiments, one plate is the same as the other plate in lateral area,thickness, shape, materials, or surface treatment.

The materials for the plates are rigid, flexible or any flexibilitybetween the two. The rigid (i.e. stiff) or flexibility is relative to agive pressing forces used in bringing the plates into the closedconfiguration.

In some embodiments, a selection of rigid or flexible plate aredetermined from the requirements of controlling a uniformity of thesample thickness at the closed configuration.

In some embodiments, at least one of the two plates are transparent (toa light). In some embodiments at least a part or several parts of oneplate or both plates are transparent. In some embodiments, the platesare non-transparent.

Plate Thickness. In some embodiments, the average thicknesses for atleast one of the pates are 2 nm or less, 10 nm or less, 100 nm or less,500 nm or less, 1000 nm or less, 2 um (micron) or less, 5 um or less, 10um or less, 20 um or less, 50 um or less, 100 um or less, 150 um orless, 200 um or less, 300 um or less, 500 um or less, 800 um or less, 1mm (millimeter) or less, 2 mm or less, 3 mm or less, or a range betweenany two of the values.

In some embodiments, the average thicknesses for at least one of theplates are at most 3 mm (millimeter), at most 5 mm, at most 10 mm, atmost 20 mm, at most 50 mm, at most 100 mm, at most 500 mm, or a rangebetween any two of the values.

In some embodiments, the thickness of a plate is not uniform across theplate. Using a different plate thickness at different location can beused to control the plate bending, folding, sample thickness regulation,and others.

Plate Shape and Area. Generally, the plates can have any shapes, as longas the shape allows a compress open flow of the sample and theregulation of the sample thickness. However, in certain embodiments, aparticular shape may be advantageous. The shape of the plate can beround, elliptical, rectangles, triangles, polygons, ring-shaped, or anysuperpositions of these shapes.

In some embodiments, the two plates can have the same size or shape, ordifferent. The area of the plates depend on the application. The area ofthe plate is at most 1 mm2 (millimeter square), at most 10 mm2, at most100 mm2, at most 1 cm2 (centimeter square), at most 5 cm2, at most 10cm2, at most 100 cm2, at most 500 cm2, at most 1000 cm2, at most 5000cm2, at most 10,000 cm2, or over 10,000 cm2, or any arrange between anyof the two values. The shape of the plate can be rectangle, square,round, or others.

In certain embodiments, at least one of the plate is in the form of abelt (or strip) that has a width, thickness, and length. The width is atmost 0.1 cm (centimeter), at most 0.5 cm, at most 1 cm, at most 5 cm, atmost 10 cm, at most 50 cm, at most 100 cm, at most 500 cm, at most 1000cm, or a range between any two of the values. The length can be as longit needed. The belt can be rolled into a roll.

Plate surface flatness. In many embodiments, an inner surface of theplates are flat or significantly flat, planar. In certain embodiments,the two inner surfaces are, at the closed configuration, parallel witheach other. Flat inner surfaces facilitates a quantification and/orcontrolling of the sample thickness by simply using the predeterminedspacer height at the closed configuration. For non-flat inner surfacesof the plate, one need to know not only the spacer height, but also theexact the topology of the inner surface to quantify and/or control thesample thickness at the closed configuration. To know the surfacetopology needs additional measurements and/or corrections, which can becomplex, time consuming, and costly.

A flatness of the plate surface is relative to the final samplethickness (the final thickness is the thickness at the closedconfiguration), and is often characterized by the term of “relativesurface flatness” is the ratio of the plate surface flatness variationto the final sample thickness.

In some embodiments, the relative surface is less than 0.01%, 0.1%, lessthan 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, lessthan 20%, less than 30%, less than 50%, less than 70%, less than 80%,less than 100%, or a range between any two of these values.

Plate surface parallelness. In some embodiments, the two surfaces of theplate is significantly parallel with each other. In certain embodiments,the two surfaces of the plate is not parallel with each other.Plate flexibility. In some embodiments, a plate is flexible under thecompressing of a CROF process. In some embodiments, both plates areflexible under the compressing of a CROF process. In some embodiments, aplate is rigid and another plate is flexible under the compressing of aCROF process. In some embodiments, both plates are rigid. In someembodiments, both plate are flexible but have different flexibility.Plate optical transparency. In some embodiments, a plate is opticaltransparent. In some embodiments, both plates are optical transparent.In some embodiments, a plate is optical transparent and another plate isopaque. In some embodiments, both plates are opaque. In someembodiments, both plate are optical transparent but have differentoptical transparency. The optical transparency of a plate refers a partor the entire area of the plate.Surface wetting properties. In some embodiments, a plate has an innersurface that wets (i.e. contact angle is less 90 degree) the sample, thetransfer liquid, or both. In some embodiments, both plates have an innersurface that wets the sample, the transfer liquid, or both; either withthe same or different wettability. In some embodiments, a plate has aninner surface that wets the sample, the transfer liquid, or both; andanother plate has an inner surface that does not wet (i.e. the contactangle equal to or larger than 90 degree). The wetting of a plate innersurface refers a part or the entire area of the plate.

In some embodiments, the inner surface of the plate has other nano ormicrostructures to control a lateral flow of a sample during a CROF. Thenano or microstructures include, but not limited to, channels, pumps,and others. Nano and microstructures are also used to control thewetting properties of an inner surface.

2.3 Spacers

Spacers' Function. In present invention, the spacers are configured tohave one or any combinations of the following functions and properties:the spacers are configured to (1) control, together with the plates, thethickness of the sample or a relevant volume of the sample (Preferably,the thickness control is precise, or uniform or both, over a relevantarea); (2) allow the sample to have a compressed regulated open flow(CROF) on plate surface; (3) not take significant surface area (volume)in a given sample area (volume); (4) reduce or increase the effect ofsedimentation of particles or analytes in the sample; (5) change and/orcontrol the wetting propertied of the inner surface of the plates; (6)identify a location of the plate, a scale of size, and/or theinformation related to a plate, or (7) do any combination of the above.Spacer architectures and shapes. To achieve desired sample thicknessreduction and control, in certain embodiments, the spacers are fixed itsrespective plate. In general, the spacer can have any shape, as long asthe spacers are capable of regulating the sample thickness during a CROFprocess, but certain shapes are preferred to achieve certain functions,such as better uniformity, less overshoot in pressing, etc.

The spacer(s) is a single spacer or a plurality of spacers. (e.g. anarray). Some embodiments of a plurality of spacers is an array ofspacers (e.g. pillars), where the inter-spacer distance is periodic oraperiodic, or is periodic or aperiodic in certain areas of the plates,or has different distances in different areas of the plates.

There are two kinds of the spacers: open-spacers and enclosed-spacers.The open-spacer is the spacer that allows a sample to flow through thespacer (i.e. the sample flows around and pass the spacer. For example, apost as the spacer), and the enclosed spacer is the spacer that stop thesample flow (i.e. the sample cannot flow beyond the spacer. For example,a ring shape spacer and the sample is inside the ring). Both types ofspacers use their height to regular the final sample thickness at aclosed configuration.

In some embodiments, the spacers are open-spacers only. In someembodiments, the spacers are enclosed-spacers only. In some embodiments,the spacers are a combination of open-spacers and enclosed-spacers.

The term “pillar spacer” means that the spacer has a pillar shape andthe pillar shape refers to an object that has height and a lateral shapethat allow a sample to flow around it during a compressed open flow.

In some embodiments, the lateral shapes of the pillar spacers are theshape selected from the groups of (i) round, elliptical, rectangles,triangles, polygons, ring-shaped, star-shaped, letter-shaped (e.g.L-shaped, C-shaped, the letters from A to Z), number shaped (e.g. theshapes like 0 1, 2, 3, 4, . . . to 9); (ii) the shapes in group (i) withat least one rounded corners; (iii) the shape from group (i) withzig-zag or rough edges; and (iv) any superposition of (i), (ii) and(iii). For multiple spacers, different spacers can have differentlateral shape and size and different distance from the neighboringspacers.

In some embodiments, the spacers may be and/or may include posts,columns, beads, spheres, and/or other suitable geometries. The lateralshape and dimension (i.e., transverse to the respective plate surface)of the spacers can be anything, except, in some embodiments, thefollowing restrictions: (i) the spacer geometry will not cause asignificant error in measuring the sample thickness and volume; or (ii)the spacer geometry would not prevent the out-flowing of the samplebetween the plates (i.e. it is not in enclosed form). But in someembodiments, they require some spacers to be closed spacers to restrictthe sample flow.

In some embodiments, the shapes of the spacers have rounded corners. Forexample, a rectangle shaped spacer has one, several or all cornersrounded (like a circle rather 90 degree angle). A round corner oftenmake a fabrication of the spacer easier, and in some cases less damageto a biological material.

The sidewall of the pillars can be straight, curved, sloped, ordifferent shaped in different section of the sidewall. In someembodiments, the spacers are pillars of various lateral shapes,sidewalls, and pillar-height to pillar lateral area ratio.

In a preferred embodiment, the spacers have shapes of pillars forallowing open flow.

Spacers' materials. In the present invention, the spacers are generallymade of any material that is capable of being used to regulate, togetherwith the two plates, the thickness of a relevant volume of the sample.In some embodiments, the materials for the spacers are different fromthat for the plates. In some embodiments, the materials for the spacesare at least the same as a part of the materials for at least one plate.

The spacers are made a single material, composite materials, multiplematerials, multilayer of materials, alloys, or a combination thereof.Each of the materials for the spacers is an inorganic material, amorganic material, or a mix, wherein examples of the materials are givenin paragraphs of Mat-1 and Mat-2. In a preferred embodiment, the spacersare made in the same material as a plate used in CROF.

Spacer's mechanical strength and flexibility. In some embodiments, themechanical strength of the spacers are strong enough, so that during thecompression and at the closed configuration of the plates, the height ofthe spacers is the same or significantly same as that when the platesare in an open configuration. In some embodiments, the differences ofthe spacers between the open configuration and the closed configurationcan be characterized and predetermined.

The material for the spacers is rigid, flexible or any flexibilitybetween the two. The rigid is relative to a give pressing forces used inbringing the plates into the closed configuration: if the space does notdeform greater than 1% in its height under the pressing force, thespacer material is regarded as rigid, otherwise a flexible. When aspacer is made of material flexible, the final sample thickness at aclosed configuration still can be predetermined from the pressing forceand the mechanical property of the spacer.

Spacer inside Sample. To achieve desired sample thickness reduction andcontrol, particularly to achieve a good sample thickness uniformity, incertain embodiments, the spacers are placed inside the sample, or therelevant volume of the sample. In some embodiments, there are one ormore spacers inside the sample or the relevant volume of the sample,with a proper inter spacer distance. In certain embodiments, at leastone of the spacers is inside the sample, at least two of the spacersinside the sample or the relevant volume of the sample, or at least of“n” spacers inside the sample or the relevant volume of the sample,where “n” may be determined by a sample thickness uniformity or arequired sample flow property during a CROF.

Spacer height. In some embodiments, all spacers have the samepre-determined height. In some embodiments, spacers have differentpre-determined height. In some embodiments, spacers can be divided intogroups or regions, wherein each group or region has its own spacerheight. And in certain embodiments, the predetermined height of thespacers is an average height of the spacers. In some embodiments, thespacers have approximately the same height. In some embodiments, apercentage of number of the spacers have the same height.

The height of the spacers is selected by a desired regulated finalsample thickness and the residue sample thickness. The spacer height(the predetermined spacer height) and/or sample thickness is 3 nm orless, 10 nm or less, 50 nm or less, 100 nm or less, 200 nm or less, 500nm or less, 800 nm or less, 1000 nm or less, 1 um or less, 2 um or less,3 um or less, 5 um or less, 10 um or less, 20 um or less, 30 um or less,50 um or less, 100 um or less, 150 um or less, 200 um or less, 300 um orless, 500 um or less, 800 um or less, 1 mm or less, 2 mm or less, 4 mmor less, or a range between any two of the values.

The spacer height and/or sample thickness is between 1 nm to 100 nm inone preferred embodiment, 100 nm to 500 nm in another preferredembodiment, 500 nm to 1000 nm in a separate preferred embodiment, 1 um(i.e. 1000 nm) to 2 um in another preferred embodiment, 2 um to 3 um ina separate preferred embodiment, 3 um to 5 um in another preferredembodiment, 5 um to 10 um in a separate preferred embodiment, and 10 umto 50 um in another preferred embodiment, 50 um to 100 um in a separatepreferred embodiment.

In some embodiments, the spacer height and/or sample thickness (i) equalto or slightly larger than the minimum dimension of an analyte, or (ii)equal to or slightly larger than the maximum dimension of an analyte.The “slightly larger” means that it is about 1% to 5% larger and anynumber between the two values.

In some embodiments, the spacer height and/or sample thickness is largerthan the minimum dimension of an analyte (e.g. an analyte has ananisotropic shape), but less than the maximum dimension of the analyte.

For example, the red blood cell has a disk shape with a minim dimensionof 2 um (disk thickness) and a maximum dimension of 11 um (a diskdiameter). In an embodiment of the present invention, the spacers isselected to make the inner surface spacing of the plates in a relevantarea to be 2 um (equal to the minimum dimension) in one embodiment, 2.2um in another embodiment, or 3 (50% larger than the minimum dimension)in other embodiment, but less than the maximum dimension of the redblood cell. Such embodiment has certain advantages in blood cellcounting. In one embodiment, for red blood cell counting, by making theinner surface spacing at 2 or 3 um and any number between the twovalues, a undiluted whole blood sample is confined in the spacing, onaverage, each red blood cell (RBC) does not overlap with others,allowing an accurate counting of the red blood cells visually. (Too manyoverlaps between the RBC's can cause serious errors in counting).

In the present invention, in some embodiments, it uses the plates andthe spacers to regulate not only a thickness of a sample, but also theorientation and/or surface density of the analytes/entity in the samplewhen the plates are at the closed configuration. When the plates are ata closed configuration, a thinner thickness of the sample gives a lessthe analytes/entity per surface area (i.e. less surface concentration).

Spacer lateral dimension. For an open-spacer, the lateral dimensions canbe characterized by its lateral dimension (sometime being called width)in the x and y—two orthogonal directions. The lateral dimension of aspacer in each direction is the same or different. In some embodiments,the lateral dimension for each direction (x or y) is . . . .

In some embodiments, the ratio of the lateral dimensions of x to ydirection is 1, 1.5, 2, 5, 10, 100, 500, 1000, 10,000, or a rangebetween any two of the value. In some embodiments, a different ratio isused to regulate the sample flow direction; the larger the ratio, theflow is along one direction (larger size direction).

In some embodiments, the different lateral dimensions of the spacers inx and y direction are used as (a) using the spacers as scale-markers toindicate the orientation of the plates, (b) using the spacers to createmore sample flow in a preferred direction, or both.

In a preferred embodiment, the period, width, and height.

In some embodiments, all spacers have the same shape and dimensions. Insome embodiments, each spacers have different lateral dimensions.

For enclosed-spacers, in some embodiments, the inner lateral shape andsize are selected based on the total volume of a sample to be enclosedby the enclosed spacer(s), wherein the volume size has been described inthe present disclosure; and in certain embodiments, the outer lateralshape and size are selected based on the needed strength to support thepressure of the liquid against the spacer and the compress pressure thatpresses the plates.

Aspect Ratio of Height to the Average Lateral Dimension of PillarSpacer.

In certain embodiments, the aspect ratio of the height to the averagelateral dimension of the pillar spacer is 100,000, 10,000, 1,000, 100,10, 1, 0.1, 0.01, 0.001, 0.0001, 0, 00001, or a range between any two ofthe values.

Spacer height precisions. The spacer height should be controlledprecisely. The relative precision of the spacer (i.e. the ratio of thedeviation to the desired spacer height) is 0.001% or less, 0.01% orless, 0.1% or less; 0.5% or less, 1% or less, 2% or less, 5% or less, 8%or less, 10% or less, 15% or less, 20% or less, 30% or less, 40% orless, 50% or less, 60% or less, 70% or less, 80% or less, 90% or less,99.9% or less, or a range between any of the values.Inter-spacer distance. The spacers can be a single spacer or a pluralityof spacers on the plate or in a relevant area of the sample. In someembodiments, the spacers on the plates are configured and/or arranged inan array form, and the array is a periodic, non-periodic array orperiodic in some locations of the plate while non-periodic in otherlocations.

In some embodiments, the periodic array of the spacers has a lattice ofsquare, rectangle, triangle, hexagon, polygon, or any combinations ofthereof, where a combination means that different locations of a platehas different spacer lattices.

In some embodiments, the inter-spacer distance of a spacer array isperiodic (i.e. uniform inter-spacer distance) in at least one directionof the array. In some embodiments, the inter-spacer distance isconfigured to improve the uniformity between the plate spacing at aclosed configuration.

The distance between neighboring spacers (i.e. the inter-spacerdistance) is 1 um or less, 5 um or less, 10 um or less, 20 um or less,30 um or less, 40 um or less, 50 um or less, 60 um or less, 70 um orless, 80 um or less, 90 um or less, 100 um or less, 200 um or less, 300um or less, 400 um or less, or a range between any two of the values.

In certain embodiments, the inter-spacer distance is at 400 or less, 500or less, 1 mm or less, 2 mm or less, 3 mm or less, 5 mm or less, 7 mm orless, 10 mm or less, or any range between the values. In certainembodiments, the inter-spacer distance is a 10 mm or less, 20 mm orless, 30 mm or less, 50 mm or less, 70 mm or less, 100 mm or less, orany range between the values.

The distance between neighboring spacers (i.e. the inter-spacerdistance) is selected so that for a given properties of the plates and asample, at the closed-configuration of the plates, the sample thicknessvariation between two neighboring spacers is, in some embodiments, atmost 0.5%, 1%, 5%, 10%, 20%, 30%, 50%, 80%, or any range between thevalues; or in certain embodiments, at most 80%, 100%, 200%, 400%, or arange between any two of the values.

Clearly, for maintaining a given sample thickness variation between twoneighboring spacers, when a more flexible plate is used, a closerinter-spacer distance is needed.

-   -   Specify the accuracy of the inter spacer distance.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 2 to 4 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 1 um to 100 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 2 to 4 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 100 um to 250 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 4 to 50 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 1 um to 100 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 4 to 50 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 100 um to 250 um.

The period of spacer array is between 1 nm to 100 nm in one preferredembodiment, 100 nm to 500 nm in another preferred embodiment, 500 nm to1000 nm in a separate preferred embodiment, 1 um (i.e. 1000 nm) to 2 umin another preferred embodiment, 2 um to 3 um in a separate preferredembodiment, 3 um to 5 um in another preferred embodiment, 5 um to 10 umin a separate preferred embodiment, and 10 um to 50 um in anotherpreferred embodiment, 50 um to 100 um in a separate preferredembodiment, 100 um to 175 um in a separate preferred embodiment, and 175um to 300 um in a separate preferred embodiment.

Spacer density. The spacers are arranged on the respective plates at asurface density of greater than one per um², greater than one per 10um², greater than one per 100 um², greater than one per 500 um², greaterthan one per 1000 um², greater than one per 5000 um², greater than oneper 0.01 mm², greater than one per 0.1 mm², greater than one per 1 mm²,greater than one per 5 mm², greater than one per 10 mm², greater thanone per 100 mm², greater than one per 1000 mm², greater than one per10000 mm², or a range between any two of the values.

(3) the spacers are configured to not take significant surface area(volume) in a given sample area (volume);

Ratio of spacer volume to sample volume. In many embodiments, the ratioof the spacer volume (i.e. the volume of the spacer) to sample volume(i.e. the volume of the sample), and/or the ratio of the volume of thespacers that are inside of the relevant volume of the sample to therelevant volume of the sample are controlled for achieving certainadvantages. The advantages include, but not limited to, the uniformityof the sample thickness control, the uniformity of analytes, the sampleflow properties (i.e. flow speed, flow direction, etc.).

In certain embodiments, the ratio of the spacer volume r) to samplevolume, and/or the ratio of the volume of the spacers that are inside ofthe relevant volume of the sample to the relevant volume of the sampleis less than 100%, at most 99%, at most 70%, at most 50%, at most 30%,at most 10%, at most 5%, at most 3% at most 1%, at most 0.1%, at most0.01%, at most 0.001%, or a range between any of the values.

Spacers fixed to plates. The inter spacer distance and the orientationof the spacers, which play a key role in the present invention, arepreferably maintained during the process of bringing the plates from anopen configuration to the closed configuration, and/or are preferablypredetermined before the process from an open configuration to a closedconfigurations.

Some embodiments of the present invention is that the spacers are fixedon one of the plates before bring the plates to the closedconfiguration. The term “a spacer is fixed with its respective plate”means that the spacer is attached to a plate and the attachment ismaintained during a use of the plate. An example of “a spacer is fixedwith its respective plate” is that a spacer is monolithically made ofone piece of material of the plate, and the position of the spacerrelative to the plate surface does not change. An example of “a spaceris not fixed with its respective plate” is that a spacer is glued to aplate by an adhesive, but during a use of the plate, the adhesive cannothold the spacer at its original location on the plate surface (i.e. thespacer moves away from its original position on the plate surface).

In some embodiments, at least one of the spacers are fixed to itsrespective plate. In certain embodiments, at two spacers are fixed toits respective plates. In certain embodiments, a majority of the spacersare fixed with their respective plates. In certain embodiments, all ofthe spacers are fixed with their respective plates.

In some embodiments, a spacer is fixed to a plate monolithically.

In some embodiments, the spacers are fixed to its respective plate byone or any combination of the following methods and/or configurations:attached to, bonded to, fused to, imprinted, and etched.

The term “imprinted” means that a spacer and a plate are fixedmonolithically by imprinting (i.e. embossing) a piece of a material toform the spacer on the plate surface. The material can be single layerof a material or multiple layers of the material.

The term “etched” means that a spacer and a plate are fixedmonolithically by etching a piece of a material to form the spacer onthe plate surface. The material can be single layer of a material ormultiple layers of the material.

The term “fused to” means that a spacer and a plate are fixedmonolithically by attaching a spacer and a plate together, the originalmaterials for the spacer and the plate fused into each other, and thereis clear material boundary between the two materials after the fusion.

The term “bonded to” means that a spacer and a plate are fixedmonolithically by binding a spacer and a plate by adhesion.

The term “attached to” means that a spacer and a plate are connectedtogether.

In some embodiments, the spacers and the plate are made in the samematerials. In other embodiment, the spacers and the plate are made fromdifferent materials. In other embodiment, the spacer and the plate areformed in one piece. In other embodiment, the spacer has one end fixedto its respective plate, while the end is open for accommodatingdifferent configurations of the two plates.

In other embodiment, each of the spacers independently is at least oneof attached to, bonded to, fused to, imprinted in, and etched in therespective plate. The term “independently” means that one spacer isfixed with its respective plate by a same or a different method that isselected from the methods of attached to, bonded to, fused to, imprintedin, and etched in the respective plate.

In some embodiments, at least a distance between two spacers ispredetermined (“predetermined inter-spacer distance” means that thedistance is known when a user uses the plates).

In some embodiments of all methods and devices described herein, thereare additional spacers besides to the fixed spacers.

Specific sample thickness. In present invention, it was observed that alarger plate holding force (i.e. the force that holds the two platestogether) can be achieved by using a smaller plate spacing (for a givensample area), or a larger sample area (for a given plate-spacing), orboth.

In some embodiments, at least one of the plates is transparent in aregion encompassing the relevant area, each plate has an inner surfaceconfigured to contact the sample in the closed configuration; the innersurfaces of the plates are substantially parallel with each other, inthe closed configuration; the inner surfaces of the plates aresubstantially planar, except the locations that have the spacers; or anycombination of thereof.

2.4 Final Sample Thickness and Uniformity

In some embodiments, significantly flat is determined relative to thefinal sample thickness, and has, depending upon on embodiments andapplications, a ratio of to the sample thickness of less than 0.1%, lessthan 0.5%, less than 1%, less than 2%, less than 5%, or less than 10%,or a range between any two of these values.

In some embodiments, flatness relative to the sample thickness may beless than 0.1%, less than 0.5%, less than 1%, less than 2%, less than5%, less than 10%, less than 20%, less than 50%, or less than 100%, or arange between any two of these values.

In some embodiments, significantly flat may mean that the surfaceflatness variation itself (measured from an average thickness) is lessthan 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, orless than 10%, or a range between any two of these values. Generally,flatness relative to the plate thickness may be less than 0.1%, lessthan 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, lessthan 20%, less than 50%, or less than 100%, or a range between any twoof these values.

2.5 Spacer Fabrication Methods.

The spacers can be fabricated on a plate in a variety of ways, usinglithography, etching, embossing (nanoimprint), depositions, lift-off,fusing, or a combination of thereof. In some embodiments, the spacersare directly embossed or imprinted on the plates. In some embodiments,the spacers imprinted into a material (e.g. plastics) that is depositedon the plates. In certain embodiments, the spacers are made by directlyembossing a surface of a CROF plate. The nanoimprinting may be done byroll to roll technology using a roller imprinter, or roll to a planarnanoimprint. Such process has a great economic advantage and hencelowering the cost.

In some embodiments, the spacers are deposited on the plates. Thedeposition can be evaporation, pasting, or a lift-off. In the pasting,the spacer is fabricated first on a carrier, then the spacer istransferred from the carrier to the plate. In the lift-off, a removablematerial is first deposited on the plate and holes are created in thematerial; the hole bottom expose the plate surface and then a spacermaterial is deposited into the hole and afterwards the removablematerial is removed, leaving only the spacers on the plate surface. Insome embodiments, the spacers deposited on the plate are fused with theplate. In some embodiments, the spacer and the plates are fabricated ina single process. The single process includes imprinting (i.e.embossing, molding) or synthesis.

In some embodiments, at least two of the spacers are fixed to therespective plate by different fabrication methods, and optionallywherein the different fabrication methods include at least one of beingdeposition, bonded, fuse, imprinted, and etched.

In some embodiments, one or more of the spacers are fixed to therespective plate(s) is by a fabrication method of being bonded, beingfused, being imprinted, or being etched, or any combination of thereof.

In some embodiments, the fabrication methods for forming such monolithicspacers on the plate include a method of being bonded, being fused,being imprinted, or being etched, or any combination of thereof.

2.6 Scale-Markers

The term “scale-marker(s) refers to the scale-marker(s) that able toassist a quantification (i.e. dimension measurement) or a control of therelevant area and/or the relative volume of a sample. In someembodiments, the scale-markers are on the first plate or the secondplate, on both on plates, on one surface of the plate, on both surfacesof the plate, between the plates, near the plates, or any combination ofthereof. In some embodiments, the scale-markers are fixed on the firstplate or the second plate, on both on plates, on one surface of theplate, on both surfaces of the plate, between the plates, near theplates, or any combination of thereof. In some embodiments, thescale-markers are deposited on the first plate or the second plate, onboth on plates, on one surface of the plate, on both surfaces of theplate, between the plates, near the plates, or any combination ofthereof. In some embodiments, some of spacers are fixed and some spacersare deposited.

In some embodiments, the scale-marks are etched scale-marks, depositedmaterials, or printed materials. In certain embodiments, the materialsthat absorbing the light, reflecting light, emitting light, or anycombination of thereof.

In some embodiments, the scale-markers are a or a plurality of object(s)with known dimensions and/or known separation distances. Examples of theobjects include, not limited to, rectangles, cylinders, or circles.

In some embodiments, the scale-markers have a dimension of in the rangeof nanometers (nm), microns (um) or millimeters (mm) or other sizes.

In some embodiments, the scale-markers are a ruler, which has scalescale-marks that are configured to measure a dimension of an object. Insome embodiments, the scale-marks are in the scale of nanometer (nm),microns (um) or millimeter (mm) or other sizes. In some embodiments, thescale marks are etched scale-marks, deposited materials, or printedmaterials. In some embodiments, the materials for the scale-markers arethe materials that absorbing the light, reflecting light, scatteringlight, interfering light, diffracting light, emitting light, or anycombination of thereof.

In some embodiments, the makers are the spacers, which server dualfunctions of “regulating sample thickness” and “providing scale-markingand/or dimension scaling”. For examples, a rectangle spacer with a knowndimension or two spacers with a known separation distance can be used tomeasure a dimension related to the sample round the spacer(s). From themeasured sample dimension, one can calculate the volume of the relevantvolume of the sample.

In some embodiments, the scale-markers is configured to at leastpartially define a boundary of the relevant volume of the sample.

In some embodiments, at least one of the scale-markers is configured tohave a known dimension that is parallel to a plane of the lateral areaof the relevant volume of the sample.

In some embodiments, at least a pair of the scale-markers are separatedby a known distance that is parallel to a plane of the lateral area.

In some embodiments, the scale-markers are configured for opticaldetection.

In some embodiments, each scale-marker independently is at least one oflight absorbing, light reflecting, light scattering, light diffracting,and light emitting.

In some embodiments, the scale-markers are arranged in a regular arraywith a known lateral spacing.

In some embodiments, each scale-marker independently has a lateralprofile that is at least one of square, rectangular, polygonal, andcircular.

In some embodiments, at least one of the scale-markers is attached to,bonded to, fused to, imprinted in, and etched in one of the plates.

In some embodiments, at least one of the scale-markers is one of thespacers.

In some embodiments, some spacers also play a role of scale-marker toquantification of a relevant volume of the sample.

In certain embodiments, a binding site(s) (that immobilizes theanalytes), storage sites, or alike, serves as a scale-marker(s). In oneembodiment, the site with a known lateral dimension interacts with lightgenerating a detectable signal, that reals the known lateral dimensionof the site, hence serving a scale-marker(s).

In another embodiment, the dimension of the sites are predeterminedbefore a CROF process and the thickness of the portion of the samplesitting on the site is, when the plates are at the closed configuration,significantly smaller than the lateral average dimension of the site,then by controlling the incubation time so that, after the incubation,(1) the majority of the analytes/entity that bind to the binding sitecome from the sample volume that sites on top of the binding site, or(2) the majority of the reagent that is mixed (diffused) into the samplevolume that sites on top of the binding site come from the storage site.In these cases, the relevant volume of the sample to the binding or thereagent mixing is the volume that is approximately equal to thepredetermined site area multiplies the sample thickness at the site. Akey reason for this be possible is that, for the given incubation time,the analytes/entity in the sample volume outside the relevant volume donot have enough time to diffuse into the binding site, or the reagentson the storage site do not have enough time to diffuse into in thesample volume outside the relevant volume.

An example to illustrate the method of measuring and/or controlling therelevant area and volume by using a site with known dimension and bylimiting the incubation time is that an assay has a binding site (i.e.the area with capture agents) of 1,000 um by 1000 um on a first plate ofa CROF process (which has a surface large than the binding site); at theclosed configuration of the plates, a sample with analytes is over thebinding site, has a thickness of about 20 um (in the bind site area) andan area larger than the binding site and is incubated for a time equalto the target analyte/entity diffusion time across the sample thickness.In this case, the majority of the analytes/entity that bind to thebinding site come from the sample volume that sites on top of thebinding site, which is 1,000 um by 1000 um by 20 um=0.02 p, because theanalytes in the sample portion that is 20 um away from the binding sitedo not have time to diffuse to the binding site (statistically). In thiscase, if the signal, due to the analytes/entity captured by the bindingsite, is measured after the incubation, one can determine theanalyte/entity concentration in the relevant area and relevant volume ofthe sample from the information (provided by the binding site) of therelevant area and relevant volume. The analyte concentration isquantified by the number of analytes captured by the binding sitedivided the relevant volume.

In some embodiments, the relevant volume is approximately equal to thebinding site area times the sample thickness, and the target analyteconcentration in the sample is approximately equal to the number ofanalyte captured by the binding site divided by the relevant samplevolume. This accuracy of the method of quantification of target analytevolume gets better as the ratio of the binding site dimension to thesample thickness gets larger (assuming the incubation time is about thetarget analyte diffusion time in the sample for a distance of the samplethickness).

Spreading Times in CROF. In the present invention, in the methods andthe devices of all paragraphs that spread the sample by two plates, thetime for spreading the sample to the final thickness at a closedconfiguration is 0.001 sec or less, 0.01 sec, 0.1 sec, 1 sec, 5 sec, 10sec, 20 sec, 30 sec, 60 sec, 90 sec, 100 sec, 150 sec, 200 sec, 300 sec,500 sec, 1000 sec, or a range between any two of the values.

In the methods and the devices of all paragraphs that spread the sampleby two plates, in a preferred embodiment, the time for spreading thesample to the final thickness at a closed configuration is 0.001 sec orless, 0.01 sec, 0.1 sec, 1 sec, 3 sec, 5 sec, 10 sec, 20 sec, 30 sec, 60sec, 90 sec, 100 sec, 150 sec, or a range between any two of the values.

In the methods and the devices of all paragraphs that spread the sampleby two plates, in a preferred embodiment, the time for spreading thesample to the final thickness at a closed configuration is 0.001 sec orless, 0.01 sec, 0.1 sec, 1 sec, 3 sec, 5 sec, 10 sec, 20 sec, 30 sec, 60sec, 90 sec, or a range between any two of the values.

In the methods and the devices of all paragraphs that spread the sampleby two plates, in a preferred embodiment, the time for spreading thesample to the final thickness at a closed configuration is 0.001 sec orless, 0.01 sec, 0.1 sec, 1 sec, 3 sec, 5 sec, 10 sec, 20 sec, 30 sec, ora range between any two of the values.

In the methods and the devices of all paragraphs that spread the sampleby two plates, in a preferred embodiment, the time for spreading thesample to the final thickness at a closed configuration is 0.001 sec orless, 0.01 sec, 0.1 sec, 1 sec, 3 sec, 5 sec, 10 sec, or a range betweenany two of the values.

In the methods and the devices of all paragraphs that spread the sampleby two plates, in a preferred embodiment, the time for spreading thesample to the final thickness at a closed configuration is 0.001 sec orless, 0.01 sec, 0.1 sec, 1 sec, 3 sec, or a range between any two of thevalues.

The embodiments and any of their combinations described in the Section 3are applied to (i.e. are combined with) other embodiments in the entiredescription of the present invention.

In one preferred embodiment, the spacers are monolithically made on theX-Plate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials.

In one preferred embodiment, the spacers are monolithically made on theX-Plate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, and the thickness of theX-Plate is from 50 um to 500 um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, and the thickness of theX-Plate is from 50 um to 250 um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate and are made of the same materials, and the thickness of theX-Plate is from 50 um to 500 um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate a thin plastic film using a mold, and are made of the samematerials, and the thickness of the X-Plate is from 50 um to 250 um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, where the plastic film areeither PMMA (polymethyl methacrylate) of PS (polystyrene).

In one preferred embodiment, the spacers are monolithically made on theX-Plate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, where the plastic film areeither PMMA (polymethyl methacrylate) of PS (polystyrene) and thethickness of the X-Plate is from 50 um to 500 um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, where the plastic film areeither PMMA (polymethyl methacrylate) of PS (polystyrene) and thethickness of the X-Plate is from 50 um to 250 um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, where the plastic film areeither PMMA (polymethyl methacrylate) of PS (polystyrene), and thespacers have either a square or rectangle shape, and have the samespacer height.

In one preferred embodiment, the spacers have a square or rectangleshape (with or without round corners).

In one preferred embodiment, the spacers have square or rectanglepillars with the pillar width (spacer width in each lateral direction)between 1 um to 200 um; pillar period (i.e. spacer period) from 2um-2000 um, and pillar height (i.e. spacer height) from 1 um-100 um.

In one preferred embodiment, the spacers made of PMMA or PS have squareor rectangle pillars with the pillar width (spacer width in each lateraldirection) between 1 um to 200 um; pillar period (i.e. spacer period)from 2 um-2000 um, and pillar height (i.e. spacer height) from 1 um-100um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate and are made of plastic materials, and the spacers have squareor rectangle pillars with the pillar width (spacer width in each lateraldirection) between 1 um to 200 um; pillar period (i.e. spacer period)from 2 um-2000 um, and pillar height (i.e. spacer height) from 1 um-100um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate and are made of the same materials, and the spacers have squareor rectangle pillars with the pillar width (spacer width in each lateraldirection) between 1 um to 200 um; pillar period (i.e. spacer period)from 2 um-2000 um, and pillar height (i.e. spacer height) from 1 um-10um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate and are made of the same materials selected from PS or PMMA orother plastics, and the spacers have square or rectangle pillars withthe pillar width (spacer width in each lateral direction) between 1 umto 200 um; pillar period (i.e. spacer period) from 2 um-2000 um, andpillar height (i.e. spacer height) from 10 um-50 um.

In one preferred embodiment of a CROF device, one plate is X-Plate andthe other plate is a planar thin film, wherein the thickness of at leastone of the plates is in a range of from 10 um to 250 um; wherein thespacers are fixed on the X-Plate, and wherein the plates and the spacerscan have the same materials or different materials and are made of PMMA(polymethyl methacrylate), PS (polystyrene), or a material of similarmechanical properties as PMMA or PS.

In one preferred embodiment of a CROF device, one plate is X-Plate andthe other plate is a planar thin film, wherein the thickness of at leastone of the plates is in a range of from 250 um to 500 um; wherein thespacers are fixed on the X-Plate, and wherein the plates and the spacerscan have the same materials or different materials and are made of PMMA(polymethyl methacrylate), PS (polystyrene), or a material of similarmechanical properties as PMMA or PS.

In one preferred embodiment of a CROF device, one plate is X-Plate andthe other plate is a planar thin film, wherein the thickness of at leastone of the plates is in a range of from 10 um to 250 um; wherein thespacers are fixed on the X-Plate, and are an array of square orrectangle pillars with the pillar width (spacer width in each lateraldirection) between 1 um to 200 um; pillar period (i.e. spacer period)from 2 um-2000 um, and pillar height (i.e. spacer height) from 1 um-100um, and wherein the plates and the spacers can have the same materialsor different materials and are made of PMMA (polymethyl methacrylate),PS (polystyrene), or a material of similar mechanical properties as PMMAor PS.

The “similar” in above paragraphs means that the difference inmechanical properties within 60%.

Guard Ring. Some embodiments have a guard ring to prevent sample flowout of the plate surface. Some embodiments of the guard ring is anenclosed wall around the sample area. The wall has a height equal to thespacer height or different from the spacer height. The wall ca be asignificant distance away from the sample measurement area.

The movable plates in a CROF process may include and/or may be coupledto a hinge, a stage, or some other positioning system that is configuredto transition the plates between an open configuration and a closedconfiguration. Movable plates may be coupled together with one or morejoints in a manner that leaves an opening to access the space betweenthe plates (e.g., to insert and/or remove sample), provided that atleast one of the joints and/or at least one of the plates is flexibleenough to achieve the described open and closed configurations. Amembrane pump is not considered to be a movable plate(s).

2. Uniform Plate Spacing and Sample Thickness (U)

In many applications of a CROF process, it is desirable to improve theuniformity of the plate spacing and hence the sample thickness at theclosed configuration, particularly when the spacing is in the micronand/or nanoscale. A good uniformity can improve the uniformity of anassay. The present invention provides the means to improve theuniformity.

The factors that can degrade the uniformity of the plate spacing in CROFinclude (a) a local bending of a plate, (b) a non-flatness of the innersurface of a plate, and (c) dusts. The smaller the final plate spacing,the worse effects these factors become.

To improve the spacing (hence sample thickness) uniformity, the presentinvention uses certain design in the plates (mechanical strength,thickness, etc.), spacer size, number of spacers, layout of the spacers,inter spacer spacing, the precision of spacer height, among other thingsto overcome the factors that cause a non-uniformity.

Inner Surface Smoothness 3.1 Use of Inter Spacer Distance to AchieveUniform Sample Thickness for a Flexible Plate

It is desirable, in some applications, to have one or both of CROFplates flexible. However, as illustrated in FIG. 5 a , for a flexibleplate (e.g. a plastic thin film), if the inter-spacer distance is toolarge, during a CROF process, the flexibility of the plate(s) can lead alocal bending (e.g. sag, namely bending inward) of the plate at thelocations that are between the two neighboring spacers, leading to apoor sample thickness uniformity. A poor sample thickness uniformity hasmany disadvantages, such as large errors in determining the samplevolume and/or analytes concentration, variation of the incubation time,etc.

One embodiment of the present invention provides a solution that reducea local bending and hence the final sample thickness variation by usinga proper inter-spacer distance. As illustrated in FIG. 5 , a CROF devicehas one rigid plate with a flat sample surface and one flexible platethat has local bending between two neighboring spacers, if the interspacer distance is too large (FIG. 5 a ). To reduce the local bending,the inter spacer distance is set to be equal or smaller the criticalbending span of the flexible plate (FIG. 5 b ). When both plates areflexible, the inter spacer distance should less than the smallest of thecritical bending span of the two plates.

U1. A method for uniformly regulating a thickness of a relevant volumeof a sample using two plates, comprising:

-   -   (a) obtaining a sample, wherein a thickness of a relevant volume        of the sample is to be regulated;    -   (b) obtaining two plates that are movable relative to each other        into different configurations; wherein one or both plates are        flexible; and wherein one or both of the plates comprise        spacers, the spacers have a predetermined inter-spacer distance        and height, and each of the spacers is fixed with its respective        plate;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are either partially or completely separated apart and        the spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and the relevant        volume of the sample are between the plates, the thickness of        the relevant volume of the sample is regulated by the plates and        the spacers;        -   wherein for the given plates, the spacers are configured to            make the thickness of the relevant volume of the sample            having a variation over a given area less than a            predetermined value; and wherein the relevant volume is a            portion or an entire volume of the sample.

In the method of paragraph U1, the configuration of the spacerscomprises selecting a proper inter spacer distance. In some embodiments,the inter spacer distance is selected, so that for an allowed samplethickness variation, given two plate, and a compression method, thebending of the two plates, under the compression method, is equal to orless than the allowed sample thickness variation. The regulated samplethickness at the closed configuration can be thinner than the maximumthickness of the sample when the plates are in the open configuration

U2. A device for regulating a thickness of a relevant volume of asample, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein one or both of the plates are flexible, and wherein one        or both of the plates comprise spacers, the spacers have a        predetermined inter-spacer distance and height, and each of the        spacers is fixed with its respective plate;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the relevant volume of the        sample are between the plates, the thickness of the relevant        volume of the sample is regulated by the plates and the spacers;    -   wherein for the given plates, the spacers are configured to make        the thickness of the relevant volume of the sample having a        thickness variation over an area less than a predetermined        value; and wherein the relevant volume is a portion or an entire        volume of the sample.

In the device of paragraph U2, the configuration of the spacers andplates comprises selecting a proper inter spacer distance. In someembodiments, the inter spacer distance is selected, so that for anallowed sample thickness variation, given two plate, and a compressionmethod, the bending of the two plates, under the compression method, isequal to or less than the allowed sample thickness variation. Theregulated sample thickness at the closed configuration can be thinnerthan the maximum thickness of the sample when the plates are in the openconfiguration

In some embodiments, small interspace spacing also allow to use flexiblethin films (e.g. Plastic file of 100 um thick) by making theinter-spacer distance less than the bending f the plate between twospacers.

In some embodiments for having a uniform sample thickness over a largearea at a closed configuration, for a given allowed maximum bending ofthe flexible plate, the ratio of inter spacer distance to the criticalbending span of the plate is at most 0.001%, at most 0.001%, at most0.001%, at most 0.01%, at most 0.1%, at most 1%, at most 10%, at most20%, at most 50%, at most 70%, at most 100%, or a range between any twoof the values.

3.2 Use of Flexible Plate(s) and Spacers to Overcome the Effects of Dustin CROF

One problem that needs to be overcome in a CROF process is that a dustwith a thickness larger than a spacer height can destroy the regulationof the spacers to achieve an intended final plate spacing (hence thesample final thickness) (illustrated in FIG. 6 a ). When two rigidplates are used, one such dust would can destroy the spacer regulationover the entire plate area.

Certain embodiments of the present invention solve the problem by usinga proper flexible plate(s) and inter spacer distance to limit the effectof the dust in a small area around the dust, while allowing the areaoutside the small area to have a final plate spacing and samplethickness set (regulated) by the spacers).

For example, FIG. 6 b illustrates that, to overcome the effects of thedust, one flexible plate with a proper flexibility is used to limit thedust area, and it is used together with a rigid plate that has fixedspacers. FIG. 6 c shows another embodiment of reducing the dust effect,where the spacers are fixed on the flexible plate. Clearly, anothersolution is to make both plate flexible.

The proper flexibility of the plates to minimize the effects of the dustin a CROF process can be selected from the thickness and the mechanicalproperty of the plate. Based on the test illustrated in an Examplepreferred embodiments are following.

U3. A method for minimizing the effects of a dust on regulating athickness of a relevant volume of a sample, comprising:

-   -   (a) obtaining a sample, wherein a thickness of a relevant volume        of the sample is to be regulated;    -   (b) obtaining two plates that are movable relative to each other        into different configurations; wherein one or both plates are        flexible; and wherein one or both of the plates comprise        spacers, the spacers have a predetermined inter-spacer distance        and height, and each of the spacers is fixed with its respective        plate;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are either partially or completely separated apart and        the spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers, the relevant        volume of the sample, and one or a plurality of dusts of a        thickness larger than the spacer height are between the plates,        the thickness of the relevant volume of the sample is regulated        by the plates and the spacers;    -   wherein the spacers and plates are configured to minimize the        area between the two plates that is affected by the dust;        wherein the area affected by the dust is the area where the dust        prevents the spacers to regulate the final spacing between the        plates in the area at a closed configuration of the plates in        the same way as if there is no dust; and wherein the relevant        volume is a portion or an entire volume of the sample.

In the method of paragraph U3, the configuration of the spacers andplates for minimizing the dust effect area comprises selecting a properthickness and mechanical property of the flexible plate.

In some embodiments, the inter spacer distance is selected, so that foran allowed sample thickness variation, given two plate, and acompression method, the bending of the two plates, under the compressionmethod, is equal to or less than the allowed sample thickness variation.The regulated sample thickness at the closed configuration can bethinner than the maximum thickness of the sample when the plates are inthe open configuration.

Specify the Flexibility of the Plate.

U4. A device for minimizing the effects of a dust on regulating athickness of a relevant volume of a sample, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations and that each plate has        a sample contact surface that contact a sample, wherein one or        both of the plates are flexible;    -   spacers on the sample contacting surface of one or both of the        plates, wherein the spacers have a predetermined inter-spacer        distance and height, and each of the spacers is fixed with its        respective plate;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers, the relevant volume of the        sample, and one or a plurality of dusts of a thickness larger        than the spacer height are between the plates, the thickness of        the relevant volume of the sample is regulated by the plates and        the spacers; and    -   wherein the spacers and plates are configured to minimize the        area between the two plates that is affected by the dust;        wherein the area affected by the dust is the area of the inner        surface of the plates where the plates and the spacers no longer        be able to regulate the sample thickness as the area that has no        dust; and wherein the relevant volume is a portion or an entire        volume of the sample.

3.3 Use of Spacers to Reducing the Effects of Surface FlatnessVariation.

In reality, no surface of plate is perfectly flat. As illustrated inFIG. 7 a , in CROF, a surface flatness variation can be significantlylarge compared with a desired sample thickness, which can causes largeerrors in determining a sample thickness. As the final sample thicknessin CROF become very thin (e.g. in micro or nanometer arrange), a surfaceflatness variation can increasingly cause significant errors.

A surface flatness variation can be characterized by the surfaceflatness variation distance of a plate, λ, is the distance from a localmaximum of a surface height to a neighboring local minimum (illustratedin FIG. 7 b ).

The present invention provides the means that make the variation of thefinal sample thickness at the closed configuration of a CROF processsmaller than the surface flatness variation on the sample surface of theplates that was existed when the plates in an open configuration. A keyapproach in the present invention for achieving a uniform final samplethickness is to use a flexible plate, a proper inter-spacer distance,and proper compressing force (illustrated in FIGS. 7 c and d ).

Considering the case where one rigid plate and a flexible plate are usedin a CROF process, at the open configuration of the plates, the samplesurface of the rigid plate has a good flatness, but the sample surfaceof the flexible plate has a significant surface flatness variation (i.e.significant compared to the intended final sample thickness), asillustrated in FIGS. 7 a and b . The present invention corrects theinitial flatness variation of the sample surface at an openconfiguration (e.g. making the flatness variation smaller) by using (i)an inter spacer distance that is less than the initial surface flatnessvariation distance; (ii) a proper compression force and/or a propercapillary force between the sample and the plates at the closedconfiguration to deform the flexible plate; and (iii) a properflexibility of the flexible plate, so that, at a final configuration ofthe plates, the sample surface of the flexible plate deforms and followsthe contour of the flat surface of the rigid plate (FIG. 7 c ).Furthermore, to reduce the final sample thickness variation, theinter-spacer distance should also be smaller than the critical bendingspan of the flexible plate as well.

The above method of correcting surface flatness variation also works forthe cases (a) the rigid plate has an initial significant sample surfaceflatness variation while the flexible plate has a smooth sample surface,(b) both the flexible plate and the rigid plate have significantflatness variation on their prospective sample surface, and (c) bothplates are flexible and the sample surface(s) of one or both plate(s)has significant surface flatness variation (FIG. 7 d ).

U5. A method for reducing the effect of surface flatness variation of aplate on the uniformity of the final thickness of a relevant volume of asample in a CROF process, comprising:

-   -   (a) obtaining a sample, wherein a thickness of a relevant volume        of the sample is to be regulated;    -   (b) obtaining two plates that are movable relative to each other        into different configurations; wherein one or both plates are        flexible; wherein one or both plates have a surface flatness        variation, and wherein one or both of the plates comprise        spacers, the spacers have a predetermined height, and each of        the spacers is fixed with its respective plate;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are either partially or completely separated apart and        the spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and the relevant        volume of the sample are between the plates, the thickness of        the relevant volume of the sample is regulated by the plates and        the spacers;    -   wherein the spacers and plates are configured to make the        thickness variation of the relevant volume of the sample at the        closed configuration is less than the surface flatness variation        of the plate(s) at the open configuration, and wherein the        relevant volume is a portion or an entire volume of the sample.

U6. A device for reducing the effect of surface flatness variation of aplate on the uniformity of regulating a thickness of a relevant volumeof a sample, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations, wherein one or both of        the plates are flexible, and one or both plates has a surface        flatness variation;    -   spacers that are fixed on one or both of the plates and have a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the relevant volume of the        sample are between the plates, the thickness of the relevant        volume of the sample is regulated by the plates and the spacers;    -   wherein the spacers and plates are configured to make the        thickness variation of the relevant volume of the sample at the        closed configuration is less than the surface flatness variation        of the plate(s) at the open configuration, and wherein the        relevant volume is a portion or an entire volume of the sample,        and the average dimension of the relevant volume is larger than        that the surface flatness variation of the plate at the open        configuration.

In the method of paragraph U5 and the device of paragraph U6, theconfiguration of the spacers and plates to reduce the effect of surfaceflatness variation of a plate on the uniformity of the final thicknessof a relevant volume of a sample comprises using a proper inter spacerdistance (IDS). One preferred embodiment is that the IDS is equal to orless than the initial surface flatness variation distance of a plate atan open configuration.

In the method and the device of paragraphs U5 and U6, in someembodiments, (1) the spacers are inside the sample at the closedconfiguration, (2) the spacers are fixed with respective plates, (3)Short inter-spacer distance, or (4) any combinations of thereof.

In the methods and the devices in the paragraphs of U1 to U8, theconfiguration of the spacers and plates that make the thickness of therelevant volume of the sample uniform has an embodiment described above.In some embodiments, the predetermined inter-spacer distance isconfigured to limit a local bending of the plates between two spacers,and wherein the relevant volume is a portion or an entire volume of thesample.

This include the cases that one or both of the plate are flexible andvarious different flexibility. (e.g. 100 um thick of PMMA or PS).

In one preferred embodiment, one plate is PMMA. In one preferredembodiment, one plate (first plate) is a glass of a thickness of 0.5 to1.5 mm thick and does not have any spacer, and the other plate (second)plate is a PMMA film of 175 um thick and has a spacer array, wherein thespacer are pillars with a rectangle shape (a dimension of 40 um inx-direction and 30 um in y-direction) with round corners and a period of120 um in x-direction and 110 um in y-direction (leading to theinter-spacer spacing of 80 um in both x and y directions).

In the methods and the devices of Section 3, some embodiments for thespacers inside of the sample at the closed configuration, the spacers'materials and the plates are the embodiments of Section 2.

In the methods and the devices of paragraphs U1-6, in some embodiments,the ratio of pillar width (or lateral average dimension) to pillarheight is 0.01 or larger, 0.1 or larger, 1 or larger, 1.5 or larger, 2or larger, 3 or larger, 5 or larger, 10 or larger, 50 or larger, 100 orlarger, or a range between any two of the values.

In the methods and the devices of paragraphs U1-6, in a preferredembodiment, the ratio of pillar width (or lateral average dimension) topillar height is 1, 1.5, 2, 10, 20, 30, 50, 100, or a range between anytwo of the values.

In the methods and the devices of paragraphs U1-6, in some embodiments,the ratio of pillar period to pillar width (or lateral averagedimension) is 1.01, 1.1, 1.2, 1.5, 1.7, 2, 3, 5, 7, 10, 20, 50, 100,500, 1000, 10,000, or a range between any two of the values.

In the methods and the devices of paragraphs U1-6, in a preferredembodiment, the ratio of pillar period to pillar width (or lateralaverage dimension) is 1.2, 1.5, 1.7, 2, 3, 5, 7, 10, 20, 30, or a rangebetween any two of the values.

In the methods and the devices of paragraphs U1-6, in a preferredembodiment, the ratio of pillar period to pillar width (or lateralaverage dimension) is 1.2, 1.5, 1.7, 2, 3, 5, 7, 10, or a range betweenany two of the values.

c) For example, in blood cell counting application, preferred X-Platepillar height is between 1 um to 5 um, pillar width is between 2 um to30 um, pillar period is between 4 um to 300 um.d) For example, in immunoassay application, preferred X-Plate pillarheight is between 5 um to 50 um, pillar width is between 10 um to 250um, pillar period is between 20 um to 2500 um.

The embodiments and any of their combinations described in the Section 3are applied to (i.e. are combined with) other embodiments in the entiredescription of the present invention.

In some embodiments, other factors are also used to control the samplethickness uniformity, these factors include, but not limited to, thesample area, the plate mechanical properties, the final sample thicknessat the closed configuration, and the plate surface wetting properties.

Below are some preferred embodiments for the methods and the devices inthe Section 1 and the rest of the disclosures.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 2 to 4 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 1 um to 100 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 2 to 4 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 100 um to 250 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 4 to 50 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 1 um to 100 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 4 to 50 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 100 um to 250 um.

The period of spacer array is between 1 nm to 100 nm in one preferredembodiment, 100 nm to 500 nm in another preferred embodiment, 500 nm to1000 nm in a separate preferred embodiment, 1 um (i.e. 1000 nm) to 2 umin another preferred embodiment, 2 um to 3 um in a separate preferredembodiment, 3 um to 5 um in another preferred embodiment, 5 um to 10 umin a separate preferred embodiment, and 10 um to 50 um in anotherpreferred embodiment, 50 um to 100 um in a separate preferredembodiment, 100 um to 175 um in a separate preferred embodiment, and 175um to 300 um in a separate preferred embodiment, and.

4 Sample and Deposition

In the present invention of the methods and devices that use a CROFprocess, the sample is deposited by several methods or a combination ofthe methods. In one embodiment of the deposition, the sample isdeposited on only one plate. In certain embodiments, the sample isdeposited on both plates (i.e. the first and the second plate).

The sample is deposited when the plates are at an open configuration. Insome embodiments, the first plate and the second plate are wellseparated from each other during the sample deposition, so that thesample is easily deposited onto one plate without a hindrance of anotherplate. For example, the first plate and the second plate can be faraway, so that the sample is directly dropped onto the first plate or thesecond plate, as if the other plate does not exist. In certainembodiments of the sample deposition, the first plate and the secondplate are separated with a distance from each other at an openingconfiguration of the plates, then the sample is deposited on the plates(e.g. by lateral flow or other dropping methods). In certain embodimentthe two plates have one side (e.g. edge) connected together during alloperations of the plates (FIG. 30 ); and an opening and a closing of thetwo plates similar to opening and closing a book.

The deposition of the sample can be a single drop or multiple drops. Themultiple drops can be at one location or multiple locations of eitherone plate or both plates. The droplets can be well separated from eachother, connected, or a combination of thereof.

In some embodiments, a sample comprises more than one materials, and thematerials are deposited together or separately. The materials aredeposited separately either in parallel or sequence.

The deposition of the sample to the plates (i.e. the first plate and thesecond plate) can be performed using a device or directly from testsubject to the plates. In some embodiments, a sample is deposited usinga device. The device include, but not limited to, pipettes, needle,stick, swab, tube, jet, liquid dispenser, tips, stick, inkjets,printers, spraying devices, etc. In certain embodiments, a sample isdeposited by a direct contacting between the sample at the sample sourceand a CROF plate without using any devices (i.e. bring the sample andthe plate together to make a contact between the two). This is termed“direct sample deposition”.

Examples of a direct sample deposition of a sample to a plate(s) are (a)a direct contact of between pricked finger (or other body parts) and aplate, (b) spitting saliva onto the plate(s), (c) taking a tear in humaneyes by a direct contact between the tear and the plate(s), (d) a directcontact between the sweat and the plate(s), and (e) a direct breathingonto the plate(s) to deposit a breath, etc. Such direct depositionmethod can be used for both human and animals.

In some embodiments, both a direct and indirect (through a device)sample deposition are used.

In present invention, the volume of the sample that is deposited on theplate or the plates (“sample volume”) is at most 0.001 pL (pico liter),at most 0.01 pL, at most 0.1 pL, at most 1 pL, at most 10 pL, at most100 pL, at most 1 nL (nano liter), at most 10 nL, at most 100 nL, atmost 1 uL (micro liter), at most 10 uL, at most 100 uL, at most 1 mL(milliliter), at most 10 mL, or a range of any two of these values.

In some embodiments, the depositing of a sample comprise the steps of(a) put a sample on one or both of the plates, and (b) spreading thesample using a means other than the second plate compression in a CROFprocess. The means of spreading the sample include using another device(e.g. stick, blade), air blow, or others.

Sample Deformation.

During a CROF process, in some embodiments, the samples behaveapproximately like an incompressible liquid (which refers a liquid thatmaintains a constant volume under a shape deformation), therefore achange in the sample thickness would lead to the change in the samplearea. In some embodiments, the samples behave like a compressibleliquid, yet their lateral area still expand when their thickness isreduced during a CROF process. In certain embodiments, the sample areliquid, gel, or soft-solids, as long as that, during a CROF process,their lateral area expands when their thickness is reduced.

In the of the present invention disclosed, “facing the first plate andthe second plate” is a process that manipulates the position andorientation of the first plate or the second plate or both, so that thesample is between the inner surfaces of the first plate and the secondplate. In some embodiments, the action of “facing the first plate andthe second plate” is performed by human hands, human hands with certaindevices, or automatic devices without human hands.

In some embodiments, the thickness is at most 1 mm, at most 100 μm, atmost 20 μm, at most 10 μm, or at most 2 μm. In some embodiments, thethickness is at least 0.1 μm. In some embodiments, further comprisingmeasuring the thickness.

In some embodiments, a variation of the thickness of the relevant volumeof the sample is at most 300%, at most 100%, at most 30%, at most 10%,at most 3%, at most 1%, at most 0.3%, or at most 0.1% of an effectivediameter of the relevant area

In some embodiments, the thickness is at least partially determined bythe predetermined height.

5. Analytes, Entity, Binding Site, Storage Site, and Transfer Media

In present invention, the entity include, but not limited to, one of aprotein, an amino acid, a nucleic acid, a lipid, a carbohydrate, ametabolite, a cell, or a nanoparticle.

In some embodiments, the binding site includes a binding partnerconfigured to bind to the respective entity.

In some embodiments, the binding site includes an entity bound to thebinding site.

In some embodiments, the placing the sample includes placing the samplewithin the binding site.

In some embodiments, the reagent includes at least one of a protein, anamino acid, a nucleic acid, a lipid, a carbohydrate, and a metabolite.

In certain embodiments, the storage site includes dried reagent.

In some embodiments, the storage site includes reagent configured to bereleased from the storage site upon contact with the sample.

In some embodiments, the first storage site and the second storage siteare in a common storage site.

In some embodiments, the transfer media is a sample. In someembodiments, the transfer media is a liquid, wherein the reagent or theentity can be dissolved and diffuse in the liquid.

In some embodiments, a plate has multiple storage sites. In anotherembodiment, one storage site has multiple reagent.

Different release time. In some embodiments, a plate has multiplestorage sites on different locations of the plate or one storage sitestores multiple reagent, and upon in touch with the sample by thestorage sites, the reagents are released but released at different timefor different reagents on the same storage site or reagents on differentstorage sites.

In some embodiments, the first reagent is configured to be released fromthe first storage site upon contact with the sample in a first averagerelease time and the second reagent is configured to be released fromthe second storage site upon contact with the sample in a second averagerelease time, and wherein the first average release time is less thanthe second average release time.

In some embodiments, the first reagent is configured to be released fromthe first storage site upon contact with the sample and wherein thesecond reagent is a bound reagent.

In some embodiments, the depositing includes binding at least one of thereagents to the respective plate.

In some embodiments, the contacting includes releasing at least one ofthe reagents from the respective plate.

In some embodiments, the depositing includes depositing a first reagentand a second reagent, and wherein the contacting includes releasing thefirst reagent before the second reagent.

In some embodiments, at least one of the plates comprises a storage sitethat includes a reagent that is to be added to the relevant volume ofthe sample. In some embodiments, wherein the reagent includes at leastone of a protein, an amino acid, a nucleic acid, a lipid, acarbohydrate, and a metabolite.

In some embodiments, the storage site includes dried reagent.

In some embodiments, the storage site includes reagent configured to bereleased from the storage site upon contact with the sample.

In some embodiments, the storage site is a first storage site and thereagent is a first reagent, wherein the device includes a second storagesite including a second reagent that is to be added into the relevantvolume of the sample, wherein the second storage site is on one of theplates.

In some embodiments, the first storage site and the second storage siteare in a common storage site.

In some embodiments, the first reagent is configured to be released fromthe first storage site upon contact with the sample in a first averagerelease time and the second reagent is configured to be released fromthe second storage site upon contact with the sample in a second averagerelease time, and wherein the first average release time is less thanthe second average release time.

In some embodiments, at least one of the reagents is dried on therespective plate.

In some embodiments of a kit, at least one of the reagents is bound tothe respective plate.

In some embodiments of a kit, at least one of the reagents is configuredto be released from the respective plate upon contact with the sample.

In some embodiments of a kit, a first reagent is on one or both of theplates and a second reagent is on one or both of the plates, wherein thefirst reagent is configured to be released from the respective plateupon contact with the sample in a first average release time and thesecond reagent is configured to be released from the respective plateupon contact with the sample in a second average release time, andwherein the first average release time is less than the second averagerelease time.

In some embodiments of the devices, the storage site is a first storagesite and the reagent is a first reagent, wherein the device includes asecond storage site including a second reagent that is to be added intothe relevant volume of the sample, wherein the second storage site is onone of the plates.

6. Locally Binding or Mixing in a Portion of a Sample (P)

In some applications, it is desirable to have a binding site to capture(i.e. bind) the analytes only in a portion of a sample, not in theentire sample. It is also desirable in some cases that a reagent isadded (i.e. mixed) into a port of a sample, not the entire sample. It isoften desirable that there is no fluidic separation between the portionof the sample and the rest of the sample. Such requirements arepreferable or necessary in certain multiplexed detections.

The present invention offers a solution to the above requirements byusing a CROF method and device to reshape a sample into a ultra-thinfilm of a thickness, that is smaller than the lateral dimension of theportion of the sample, wherein only an analyte inside that portion ofthe sample will be captured, or only the portion of the sample will bemixed with a reagent. The working principle for such approach is thatwhen the thickness of the sample is smaller than the lateral dimensionof the portion of the sample, a capture of an analyte by a surface or amixing of reagent placed on a surface can be primarily limited by thediffusion of the analytes and the reagent in the thickness direction,where the diffusion in the lateral diffusion is relativelyinsignificant. For example, if a sample is reshaped in to a thin film of5 um thick, if the portion of the sample that an analyte should becaptured or a reagent should be mixed has a lateral dimension of 5 mm by5 mm, and if the diffusion time of analyte or reagent across the 5 um is10 sec, then the lateral diffusion of the analyte or the reagent acrossthe 5 mm distance is 1,000,000 sec (since the diffusion time isproportional to the square of the diffusion distance). This means thatby selecting a proper ratio of the lateral dimension of the interestedportion of the sample to the sample thickness, in certain time interval,the analytes captured primarily come from the sample portion interested,or the regent is mixed primarily into the portion of the sample ofinterest.

6.1 Locally Binding of Entity in a Portion of a Sample to a Surface (P:Volume to Surface)

P1. A method for locally bind target entities in a relevant volume of asample to a binding site on a surface, comprising:

-   -   (i) perform the steps of (a) to (d) in the method of paragraph        X1, wherein the sample thickness at the closed configuration is        significantly less than the average linear dimension of the        binding site; and wherein the relevant volume is the volume of        the sample that sits on the binding site when the plates are in        the closed configuration;    -   (ii) after (i) and while the plates are in the closed        configuration, either:        -   (1) incubating the sample for a relevant time length and            then stopping the incubation; or        -   (2) incubating the sample for a time that is equal or longer            than the minimum of a relevant time length, and then            assessing, within a time period that is equal or less than            the maximum of the relevant length of time, the binding of            target entity to in the binding site;    -   wherein the relevant time length is:        -   i. equal to or longer than the time that it takes for the            target entity to diffuse across the thickness of the uniform            thickness layer at the closed configuration; and        -   ii. significantly shorter than the time that it takes the            target entity to laterally diffuse across the minimum            lateral dimension of the binding site;

wherein at the end of the incubation in (1) or during the assessing in(2), the majority of the target entity bound to the binding site is froma relevant volume of the sample;

wherein the incubation allows the target entity to bind to the bindingsite, and wherein the relevant volume is a portion of the sample that isabove the binding site at the closed configuration.

The method of paragraph P2, wherein the term “the thickness of arelevant volume of the sample is significantly less than the minimumaverage dimension of the binding site” means that the ratio of theminimum average dimension of the binding site to the sample thickness(termed “length to thickness ratio”) is at least 3, at least 5, at least10, at least 20, at least 50, at least 100, at least 500, at least1,000, at least 10,000, at least 100,000, or any range between thevalues. In preferred embodiments, the length to thickness ratio is atleast 3, at least 5, at least 10, at least 20, at least 50, at least100, at least 500, or any range between the values.

The method of paragraph P2, wherein the term “significantly shorter thanthe time that it takes the target entity to laterally diffuse across theminimum lateral dimension of the binding site” means that the ratio ofthe time for diffusing across the minimum lateral dimension of thebinding site to the time for diffusion across the sample thickness(termed “length to thickness diffusion time ratio”) is at least 3, atleast 10, at least 50, at least 10, at least 100, at least 1,000, atleast 10,000, at least 100,000, at least 1,00,000, or any range betweenthe values. In preferred embodiments, the length to thickness diffusiontime ratio is at least 3, at least 10, at least 50, at least 10, atleast 100, at least 1,000, at least 10,000, or any range between thevalues.

P2. A device for locally binding entity in a relevant volume of a sampleto a binding site on surface, comprising:

-   -   a first plate and a second plate, that are movable relative to        each other into different configurations,    -   wherein the first plate has, on its surface, a binding site that        has an area smaller than that of the plate and is configured to        bind target entity in a sample, wherein the target entity are        capable of diffusing in the sample, and wherein one or both of        the plates comprise spacers and each of the spacers is fixed        with its respective plate and has a predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates,    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers, the binding site, and at least a        portion of the sample are between the plates, the sample        contacts at least a part of the binding site, the thickness of a        relevant volume of the sample is regulated by the plates and the        spacers, is thinner than the maximum thickness of the sample        when the plates are in the open configuration, wherein the        relevant volume is the volume of the sample that sits on the        binding site;    -   wherein the spacer height is selected to regulate the thickness        of the relevant volume at the closed configuration to be at        least 3 times less than the average linear dimension of the        binding site.

The regulation of the thickness of the relevant volume to 3 times lessthan the average linear dimension of the binding site makes thediffusion time of the entity across the sample thickness is 9 times lessthan that across a distance equal to the average linear dimension of thebinding site. Such thickness regulation makes it possible to select anincubation time, such that the incubation results in (i) a significantnumber of target entity in the relevant volume are bound to the bindingsite and (ii) a significant number of the target entity bound to thebinding site are from the relevant volume of the sample, and wherein theincubation is a process to allow the target entity to bind to thebinding site.

For example, if the incubation time is set to be the time that equals tothe diffusion time of the entity across the thickness of the relevantvolume of the sample, then after the incubation, most of the entityinside the relevant volume are already reached the binding site andbeing bound according to the rate equation, while the entity originally(i.e. before the incubation) outside of the relevant volume can onlydiffuse into the peripheral of the relevant volume (relative smallvolume) and such volume becomes less significant, as the ratio of theaverage linear dimension of the binding site to the relevant volumethickness gets larger.

6.2 Locally Binding Entity Stored on a Plate Surface to a Binding-Siteon Other Plate Surface (Surface to Surface)

P3. A method for locally binding entity stored on a storage site of oneplate to a binding site on another plate, comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein a        surface of first plate has a binding site; and a surface of the        second plate has a storage site that comprises entity to be        bound to the binding site; wherein the area of the binding site        and the area of the reagent site is less than that of respective        plates; and wherein one or both of the plates comprise spacers        and each of the spacers is fixed with its respective plate and        has a predetermined height;    -   (b) obtaining a transfer medium, wherein the entity are capable        of being dissolving into the transfer medium and diffusing in        the transfer medium;    -   (c) depositing, when the plates are configured in an open        configuration, the transfer medium on one or both of the plates;        wherein the open configuration is a configuration in which the        two plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the transfer medium by bringing the        plates into a closed configuration, wherein, in the closed        configuration: the plates are facing each other, the spacers,        the binding site, the storage site and at least a portion of the        transfer medium are between the plates; at least a portion of        the storage site is directly facing the binding site with a        portion of the transfer medium between them, and the thickness        of a relevant volume of the transfer medium is regulated by the        plates and the spacers, is thinner than the maximum thickness of        the sample when the plates are in the open configuration, and is        significantly less than the average linear dimension of the        relevant volume in the plate surface direction; and    -   (e) after (d) and while the plates are in the closed        configuration, incubating for a time and stopping the        incubation, wherein the incubation time is selected in such that        results in a significant number of the entity bound to the        binding site are from the storage site, wherein the relevant        volume is the volume of the transfer medium that sits on the        binding site and the incubation is a process to allow the entity        to bind to the binding site.

The term of “at least a port of the storage site is directly facing thebinding site” means that the shortest distance from a point in theportion to the binding site is the same as the thickness of the relevantvolume at the closed configuration of the plates.

P4. A device for binding entity stored on a storage site of one plate toa relevant binding site on another plate, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations, wherein a surface of        first plate has a binding site; and a surface of the second        plate has a storage site that contains entity to be bound to the        binding site; wherein the area of the binding site and the area        of the storage site is less than that of respective plates; and        wherein one or both of the plates comprise spacers and each of        the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and a transfer medium is deposited on one or        both of the plates, wherein the entity on the storage site are        capable of being dissolving into the transfer medium and        diffusing in the transfer medium,    -   wherein another of the configuration is a closed configuration,        which is configured after the transfer medium deposition in the        open configuration; and in the closed configuration: the plates        are facing each other, the spacers, the binding site, the        storage site and at least a portion of the transfer medium are        between the plates; at least a portion of the storage site is        directly facing the binding site with a portion of the transfer        medium between them, and the thickness of a relevant volume of        the transfer medium is regulated by the plates and the spacers,        and is thinner than the maximum thickness of the sample when the        plates are in the open configuration;    -   wherein the relevant volume is the volume of the transfer medium        that sits on the storage site when the plates are in closed        configuration; and    -   wherein the spacer height is selected to regulate the thickness        of the relevant volume at the closed configuration to be at        least 3 times less than the average linear dimension of the        binding site.    -   wherein at least one of the spacers is inside the sample contact        area;    -   and the spacers that have a predetermined inter-spacer distance        and height.

6.3 A Method for Locally Binding Entity on Multiple Storage Sites of OnePlate to Multiple Corresponding Binding Sites on Another Plate

P5. A method for locally binding entity stored on multiple storage sitesof one plate to multiple corresponding binding sites on another plate,comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations; wherein a        surface of first plate has multiple binding sites, and a surface        of the second plate has multiple corresponding storage sites;        wherein each corresponding storage site is located in a location        on the second plate that is corresponding to the location of a        binding site, so that when the two plates are placed        face-to-face, each binding site overlaps only one storage site;        and wherein one or both of the plates comprise spacers and each        of the spacers is fixed with its respective plate and has a        predetermined height;    -   (b) obtaining a transfer medium, wherein the entity on the        storage sites are capable of being dissolving into the transfer        medium and diffusing in the transfer medium;    -   (c) depositing, when the plates are configured in an open        configuration, the transfer medium on one or both of the plates;        wherein the open configuration is a configuration in which the        two plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the transfer medium by bringing the        plates into a closed configuration, wherein, in the closed        configuration: the two plates are facing each other, the        spacers, the binding sites, the storage sites and at least a        portion of the transfer medium are between the plates, each        binding site directly faces only one corresponding storage site,        the transfer medium contacts at least a part of each of the        binding sites and a part of each of the storage sites, the        thickness of a relevant volume of the transfer medium is        regulated by the plates and the spacers, is thinner than the        maximum thickness of the transfer medium when the plates are in        the open configuration, and is significantly less than the        average linear dimension of the binding sites; and    -   (e) after (d) and while the plates are in the closed        configuration, incubating for a time and stopping the        incubation, wherein the incubation time is selected in such that        results in a significant number of the entity bound to each        binding site are from a corresponding storage site, wherein the        relevant volume is the volume of the transfer medium that sits        on the binding sites, and the incubation is a process to allow        the entity to be bound to the binding site.

In some embodiments the spacing is limited to the binding sample area.

In some embodiments of the method P5, the transfer medium is a samplewith target analyte, the binding site comprises capture agent, and theentity in the storage site is detection agent, wherein the targetanalyte binds the capture agent and the detection agent to form acapture agent-analyte-detection agent sandwich. The method P5 simplifyan assay steps and can reduce the assay time by using smaller spacerheight to have a thinner sample thickness and shorter vertical diffusiontime for both analytes and detection agents for a shorter saturationassay time.

P6. A device for locally binding entity stored on multiple storage sitesof one plate to multiple corresponding binding sites on another plate,comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein a surface of first plate has multiple binding sites, and        a surface of the second plate has multiple corresponding storage        sites; wherein each corresponding storage site is located in a        location on the second plate that is corresponding to the        location of a binding site, so that when the two plates are        placed face-to-face, each binding site overlaps only one storage        site; and wherein one or both of the plates comprise spacers and        each of the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and a transfer medium is deposited on one or        both of the plates, wherein the entity on the storage site are        capable of being dissolving into the transfer medium and        diffusing in the transfer medium,    -   wherein another of the configuration is a closed configuration,        which is configured after the transfer medium deposition in the        open configuration; and in the closed configuration: the two        plates are facing each other, the spacers, the binding sites,        the storage sites and at least a portion of the transfer medium        are between the plates, each binding site directly faces only        one corresponding storage site, the transfer medium contacts at        least a part of each of the binding sites and a part of each of        the storage sites, the thickness of a relevant volume of the        transfer medium is regulated by the plates and the spacers, and        is thinner than the maximum thickness of the transfer medium        when the plates are in the open configuration;    -   wherein the relevant volume is the volume of the transfer medium        that sits on the storage site when the plates are in closed        configuration; and    -   wherein the predetermined spacer height is selected to regulate        the thickness of the relevant volume at the closed configuration        to be significantly less than the average linear dimension of        the binding sites.

6.4 Locally Adding Reagent Stored on a Surface to a Portion of a Sample(Surface to Volume)

P7. A method for locally adding a reagent into a relevant volume of asample, comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        the first plate has, on its surface, a storage site that        contains reagents to be added into a relevant volume of a        sample, the reagents are capable of being dissolving into the        sample and diffusing in the sample, and the area of the storage        site is less than that of the plate; and wherein one or both of        the plates comprise spacers and each of the spacers is fixed        with its respective plate and has a predetermined height;    -   (b) obtaining the sample;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration; wherein, in the closed configuration:        the plates are facing each other; the spacers, the storage site,        and at least a portion of the sample are between the plates; the        sample contacts at least a portion of the storage site and        contacts the plates over an area that is larger than that of the        storage site; the thickness of a relevant volume of the sample        is regulated by the plates and the spacers, is thinner than the        maximum thickness of the sample when the plates are in the open        configuration, and is significantly less than the average linear        dimension of the relevant volume in the plate surface direction;        and    -   (e) after (d) and while the plates are in the closed        configuration, incubating for a time and stopping the        incubation, wherein the incubation time is selected in such that        results in (i) a significant number of the reagents dissolved in        the sample are contained in the relevant volume of the sample        and (ii) the reagents are in the significant part of the        relevant volume, and wherein the relevant volume is the volume        of the sample that sits on the storage site when the plates are        in closed configuration, and the incubation is a process to        allow the reagent to dissolve and diffuse in the sample.

P8. A device for locally adding a reagent stored on a plate surface intoa relevant volume of a sample, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations,    -   wherein the first plate has, on its surface, a storage site that        contains reagents to be added into a relevant volume of a        sample, the reagents are capable of being dissolving into the        sample and diffusing in the sample; and wherein one or both of        the plates comprise spacers and each of the spacers is fixed        with its respective plate and has a predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers, the storage site, and at least a        portion of the sample are between the plates, the sample        contacts at least a portion of the storage site and at least a        port of plate surface outside the storage site, the thickness of        a relevant volume of the sample is regulated by the plates and        the spacers, is thinner than the maximum thickness of the sample        when the plates are in the open configuration, and wherein the        relevant volume is the volume of the sample that sits on the        storage site when the plates are in closed configuration;    -   wherein the spacer height is selected to regulate the thickness        of the relevant volume at the closed configuration of the plates        to be at least 3 times less than the average linear dimension of        the relevant volume in the plate surface direction.

7 Formation of Capture-Analyte-Detection Sandwich on a Binding Site (W)

One aspect of the present invention is to form acapture-analyte-detection sandwich on a binding site on a solid surfacein a single step by using a CROF process and by putting the binding siteon one plate and a storage site which stores the detection agent on thecorresponding location of the other plate.

7.1 Forming Capture-Analyte-Detection Sandwich on a Binding Site in aSingle Step of Incubation (General) (W)

W1. A method for forming a capture-analyte-detection sandwich on abinding site of a plate, comprising:

-   -   (a) obtaining a sample that contains a target analyte, wherein        the target analyte is capable of diffusion in the sample;    -   (b) obtaining capture agents and obtaining detection agents,        wherein the capture agents and the detection agents (are capable        to) bind to the target analyte to form a capture agent-target        analyte-detection agent sandwich;    -   (c) obtaining a first plate and a second plate that are movable        relative to each other into different configurations; wherein        the first plates has a binding site that has the capture agents        being immobilized on the site, and the second plate has a        storage site that stores the detection agents; wherein when the        storage site is in contact with the sample, the detection agents        are capable to be dissolved into the sample and diffuse in the        sample; and wherein one or both of the plates comprise spacers        and each of the spacers is fixed with its respective plate and        has a predetermined height;    -   (d) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (e) after (d), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and a relevant        volume of the sample are between the plates, the thickness of        the relevant volume of the sample is regulated by the plates and        the spacers, and is thinner than the sample thickness when the        plates are in the open configuration, and the sample is in        contact with the binding site and the storage site; and    -   (f) after (e), while the plates are in the closed configuration,        incubating for a time to allow a formation of capture        agent-target analyte-detection agent sandwich; wherein the        relevant volume is at least a portion or an entire volume of the        sample.

W2. A device for forming a capture-analyte-detection sandwich on abinding site of a plate, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein the first plates has a binding site that has capture        agents being immobilized on the site, and the second plate has a        storage site that stores detection agents; wherein the capture        agents and the detection agents (are capable to) bind to a        target analyte in a sample to form a capture agent-target        analyte-detection agent sandwich; wherein when the storage site        is in contact with the sample, the detection agents are capable        to be dissolved into the sample and diffuse in the sample; and        wherein one or both of the plates comprise spacers and each of        the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and a relevant volume of the        sample are between the plates, the thickness of the relevant        volume of the sample is regulated by the plates and the spacers        and is thinner than the sample thickness when the plates are in        the open configuration, and the sample is in contact with the        binding site and the storage site; and    -   wherein the relevant volume is at least a portion or an entire        volume of the sample.        7.2 Forming Capture-Analyte-Detection Sandwich on a Binding Site        in a Single Step Incubation Using the Analyte that is from a        Portion of the Sample (i.e. Locally).

W3. A method for forming a capture-analyte-detection sandwich on abinding site of a plate using the analytes that are from a portion ofthe sample, comprising:

-   -   (a) obtaining a sample that contains a target analyte, wherein        the target analyte is capable of diffusion in the sample;    -   (b) obtaining capture agents and obtaining detection agents,        wherein the capture agents and the detection agents are capable        to bind to the target analyte to form a capture agent-target        analyte-detection agent sandwich;    -   (c) obtaining a first plate and a second plate that are movable        relative to each other into different configurations; wherein        the first plates has a binding site that has the capture agents        being immobilized on the site, and the second plate has a        storage site that stores the detection agents, which, when the        reagent a storage site is in contact with the sample, are        capable to be dissolved into the sample and diffuse in the        sample; and wherein one or both of the plates comprise spacers        and each of the spacers is fixed with its respective plate and        has a predetermined height;    -   (d) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (e) after (d), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers, the binding site,        and the storage site are between the plates, the binding site        and the storage site are in contact with a relevant volume of        the sample, and the thickness of the relevant volume of the        sample is regulated by the plates and the spacers and is thinner        than the sample thickness when the plates are in the open        configuration; and is significantly less than the average linear        dimension of the binding site; and    -   (f) after (e) and while the plates are in the closed        configuration, incubating for a time and stopping the        incubation, wherein the incubation time is selected in such that        results in a significant number of the capture-analyte-detection        sandwich formed at the binding site contain the analytes that        come from the relevant volume of the sample, wherein the        relevant volume is the volume of the sample that sits on the        binding site, and the incubation is a process to allow a        formation of a capture-analyte-detection sandwich.

In some embodiments the ratio of the spacing to the site dimension maybe less than 1/5.

W4. A device for forming a capture-analyte-detection sandwich on abinding site of a plate with the analytes that are from a portion of thesample, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein the first plates has a binding site that has capture        agents being immobilized on the site, and the second plate has a        storage site that stores detection agents; wherein the capture        agents and the detection agents (are capable to) bind to a        target analyte in a sample to form a capture agent-target        analyte-detection agent sandwich; wherein when the storage site        is in contact with the sample, the detection agents are capable        to be dissolved into the sample and diffuse in the sample; and        wherein one or both of the plates comprise spacers and each of        the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers, the binding site, and the        storage site are between the plates, the binding site and the        storage site are in contact with a relevant volume of the        sample, and the thickness of the relevant volume of the sample        is regulated by the plates and the spacers and is thinner than        the sample thickness when the plates are in the open        configuration; and wherein the relevant volume is the volume of        the sample that sits on the binding site; and    -   wherein the spacer height is selected to regulate the thickness        of the relevant volume at the closed configuration to be        significantly less than the average linear dimension of the        binding site.

7.3 A Method for Reducing the Time of Forming Capture-Analyte-DetectionSandwich on a Binding Site by Reducing the Diffusion Distance (W, X).

W5. A method for reducing the time of forming acapture-analyte-detection sandwich on a binding site of a plate,comprising:

-   -   (a) obtaining a sample that contains a target analyte, wherein        the target analyte is capable of diffusion in the sample;    -   (b) obtaining capture agents and obtaining detection agents,        wherein the capture agents and the detection agents are capable        to bind to the target analyte to form a capture agent-target        analyte-detection agent sandwich;    -   (c) obtaining a first plate and a second plate that are movable        relative to each other into different configurations; wherein        the first plates has a binding site that has the capture agents        being immobilized on the site, and the second plate has a        storage site that stores the detection agents, which, when the        reagent a storage site is in contact with the sample, are        capable to be dissolved into the sample and diffuse in the        sample; and wherein one or both of the plates comprise spacers        and each of the spacers is fixed with its respective plate and        has a predetermined height;    -   (d) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (e) after (d), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers, the binding site,        and the storage site are between the plates, the binding site        overlaps the storage site, the binding site and the storage site        are in contact with a relevant volume of the sample, and the        thickness of the relevant volume of the sample is regulated by        the plates and the spacers and is thinner than the sample        thickness when the plates are in the open configuration; and        thereby the reduced thickness of the sample reduces the time for        the analytes and the detection agents diffusing vertically        across the thickness of the sample, wherein the relevant volume        is at least a portion of an entire volume of the sample.        wherein the time period to allow the target entity in the        relevant volume to bind to the binding site is shorter than that        without the closed configuration.    -   the method may further comprise a wash step to remove the sample        between the plates, and the wash step is performed when the        plates are in either a closed configuration or an open        configuration.    -   The methods further comprise a read step that reads the signal        from the capture-analyte-detection sandwich immobilized on the        binding site. The read is performed either after a wash or        without any wash.        The method may further be multiplexed, as described above or        below.

W6. A device for reducing the time of forming acapture-analyte-detection sandwich on a binding site of a plate,comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein the first plates has a binding site that has capture        agents being immobilized on the site, and the second plate has a        storage site that stores detection agents; wherein the capture        agents and the detection agents (are capable to) bind to a        target analyte in a sample to form a capture agent-target        analyte-detection agent sandwich; wherein when the storage site        is in contact with the sample, the detection agents are capable        to be dissolved into the sample and diffuse in the sample; and        wherein one or both of the plates comprise spacers and each of        the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers, the binding site, and the        storage site are between the plates, the binding site overlaps        the storage site, the binding site and the storage site are in        contact with a relevant volume of the sample, and the thickness        of the relevant volume of the sample is regulated by the plates        and the spacers and is thinner than the sample thickness when        the plates are in the open configuration; and thereby the        reduced thickness of the sample reduces the time for the        analytes and the detection agents diffusing vertically across        the thickness of the sample, wherein the relevant volume is at        least a portion of an entire volume of the sample.

In these embodiments, the method may comprise attaching a capture agenta plate, wherein the attaching is done via a chemical reaction of thecapture agent with a reactive group on the plate. The other plate maycontain a patch of a dried detection reagent at a location such that,after the plates are closed, the affixed capture agent and the patch ofdetection reagent are facing each other. Next, the method may comprisecontacting a sample containing a target-analyte with the device andclosing the plates, as described above. The detection reagent dissolvesand diffuses into the sample. Since the target analyte is in solution,the target analyte will be bound by the capture agent and immobilized tothe surface of one of the plates. The detection agent can bind to thetarget analyte before or after it is bound to the capture agent. In somecases, the method may comprises removing any target-analytes that arenot bound to the capture agent, or any unbound detection reagent (e.g.,by washing the surface of a plate in binding buffer); The detectionagent may be conjugated with an optical detectable label, therebyproviding a way to detect the target analyte. After optionally removingthe detection agent that are not bound to the target-analyte, the systemcan be read, e.g., using a reading system, to read a light signal (e.g.,light at a wavelength that is in the range of 300 nm to 1200 nm) fromdetection agent that is bound to the plate. Further, as mentioned above,the detection agent may be labeled directly (in which case the detectionagent may be strongly linked to a light-emitting label prior todeposition onto one of the plates), or labeled indirectly (i.e., bybinding the detection agent to a second capture agent, e.g., a secondaryantibody that is labeled or a labeled nucleic acid, that specificallybinds to the detection agent and that is linked to a light-emittinglabel). In some embodiments, the method may comprise a blocking agent,thereby preventing non-specific binding of the capture agents tonon-target analytes. Suitable conditions for the specific binding oftarget analytes to other agents, include proper temperature, time,solution pH level, ambient light level, humidity, chemical reagentconcentration, antigen-antibody ratio, etc., are all well known orreadily derivable from the present disclosure. General methods formethods for molecular interactions between capture agents and theirbinding partners (including analytes) are well known in the art (see,e.g., Harlow et al, Antibodies: A Laboratory Manual, First Edition(1988) Cold spring Harbor, N.Y.; Ausubel, et al, Short Protocols inMolecular Biology, 3rd ed., Wiley & Sons, 1995). The methods describedabove and below are exemplary; the methods herein are not the only waysof performing an assay.

In certain embodiments, a nucleic acid capture agent can be used tocapture a protein analyte (e.g., a DNA or RNA binding protein). Inalternative embodiments, the protein capture agent (e.g., a DNA or RNAbinding protein) can be used to capture a nucleic acid analyte.

The sample may be a clinical sample derived from cells, tissues, orbodily fluids. Bodily fluids of interest include but are not limited to,amniotic fluid, aqueous humour, vitreous humour, blood (e.g., wholeblood, fractionated blood, plasma, serum, etc.), breast milk,cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime, endolymph,perilymph, feces, gastric acid, gastric juice, lymph, mucus (includingnasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleuralfluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, sweat,synovial fluid, tears, vomit, urine and exhaled condensate.

In one embodiment of this assay, a plate is contacted with a samplecontaining a target analyte (e.g., a target protein) and the plates areclosed. The sample contains, or is amended to contain, all necessaryreagents (e.g., salts and the like) conditions suitable for specificbinding. The capture agents (e.g., antibodies) and detection agentspecifically bind to a target analyte in the sample, thereby leading toa patch of labeled analyte that can be detected.

As in any embodiment, the amount of target analyte in the sample can bemeasured to provide a qualitative or quantitative measure of the amountof target analyte in the sample. In some embodiments, the magnitude ofthe signal provides a quantitative determination of the amount of targetanalyte in the sample. In some cases, the evaluation may be compared toa standard curve (e.g., of a second analyte or a spiked-in analyte) thatmay in certain cases be at a known concentration. This comparison may befacilitated by depositing capture agents at different densities (e.g.,different concentrations) and reading the signal from each patch ofcapture agent.

8 Binding and Adding Using Samples and Reagent with Small Volume (V)

It is highly desirable, in many applications, to use as small volume ofa sample or reagent as possible. However, in microfluidic channeldevices (the most popular approach today for using small samples), asignificant volume of the sample is wasted in flowing from an inlet to atesting (detection) region of the device, resulting a need to a samplevolume larger than the volume in the testing location. One aspect of thepresent invention is to significantly reduce the volume of the sample orreagent used in a testing, by depositing a tiny volume of a sample or areagent on a plate and then reshaping the volume into a thin film with asmaller thickness but larger area than before. Such reshaping alsoallows faster reaction.

8-1 Binding Target Entity in a Small Volume Sample on a Surface BindingSite by Spreading the Sample.

V1. A method for binding target entity in a sample to a binding site,comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        the first plate has, on its surface, a binding site, and wherein        one or both of the plates comprise spacers and each of the        spacers is fixed with its respective plate and has a        predetermined height;    -   (b) obtaining a sample that contains a target entity to be bound        to the binding site;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein,        in the open configuration: the two plates are partially or        completely separated apart, the spacing between the plates is        not regulated by the spacers, and the sample, as deposited,        covers either no area or a partial area of the binding site;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration; wherein, in the closed configuration:        the plates are facing each other, the spacers and a relevant        volume of the sample are between the plates, the sample contacts        more area of the binding site than that when the plates are in        the open configuration, and the thickness of the relevant volume        of the sample on the binding site is regulated by the plates and        the spacers, wherein the relevant volume is a portion or an        entire volume of the sample.

V2. A device for binding target entity in a sample to a surface bindingsite, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein the first plate has, on its surface, a binding site that        binds target entity in a sample, and wherein the binding site        has an area larger than the contact area of the sample when the        sample is deposited on only one of the plates and without        contacting the other plate;    -   wherein one or both of the plates comprise spacers and each of        the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates and covers, as deposited, either no area or a partial        area of the binding site;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the sample are between the        plates, the sample contacts more area of the binding site than        that when the plates are in the open configuration, and the        thickness of the sample on the binding site is regulated by the        plates and the spacers.        8-2 Adding Reagents into a Small Volume Sample by Spreading the        Sample

V3. A method for binding target entity in a sample to a binding site,comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        the first plate has, on its surface, a storage site that        contains the reagents to be added into the sample, and wherein        one or both of the plates comprise spacers and each of the        spacers is fixed with its respective plate and has a        predetermined height;    -   (b) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein,        in the open configuration: the two plates are partially or        completely separated apart, the spacing between the plates is        not regulated by the spacers, and the sample, as deposited,        contacts either no area or a partial area of the storage site;    -   (c) after (b), spreading the sample by bringing the plates into        a closed configuration; wherein, in the closed configuration:        the plates are facing each other, the spacers and a relevant        volume of the sample are between the plates, the sample contacts        more area of the storage site than that when the plates are in        the open configuration, and the thickness of the relevant volume        of the sample is regulated by the spacer; and wherein the        relevant volume is a portion of the sample that site on the        storage site.

V4. A device for binding target entity in a sample to a binding site,comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations,    -   wherein the first plate has, on its surface, a storage site that        contains reagents and the reagents are to be added into the        sample, and wherein one or both of the plates comprise spacers        and each of the spacers is fixed with its respective plate and        has a predetermined height;    -   wherein one or both of the plates comprise spacers and each of        the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates,    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and a relevant volume of the        sample are between the plates, the sample contacts more areas of        the storage site than that when the plates are in the open        configuration, and the thickness of the relevant volume of the        sample is regulated by the spacer; and wherein the relevant        volume is a portion of the sample that site on the storage site.

In the methods of paragraph V1 and V2 and the devices of V3 and V4, insome cases, even a sample is deposited in the binding site area or thestorage area, due to the small volume of the sample and a wettingproperty of the surface, the contact area of as-deposited sample with aplate will be less than the area of the binding site or the storagesite. Hence, a spreading, particular precisely spreading is needed.

Drops of a sample can be multiple drops, and in the closedconfiguration, the drops merged into a film with a thickness less thanthe maximum thickness.

In present invention, in the method in paragraph V1 to V7 and thedevices in paragraph of V2 to V8, the volume of the sample that isdeposited on the plate or the plates (“sample volume”) is at most 0.001pL (pico liter), at most 0.01 pL, at most 0.1 pL, at most 1 pL, at most10 pL, at most 100 pL, at most 1 nL (nano liter), at most 10 nL, at most100 nL, at most 1 uL (micro liter), at most 10 uL, at most 100 uL, atmost 1 mL (milliliter), at most 10 mL, or a range of any two of thesevalues.

9 Uniform Binding or Adding Reagents Using Uniform Sample Thickness(UAB)

For assays and chemical reactions, it is advantageous to make a thinsample thickness uniform over a significant area. The examples includebinging of entity of sample to a surface binding site, adding reagentsinto a sample, quantification a relevant volume of the sample,quantification of analytes, and others. For the methods that use twoplates to reduce and regulate a thickness of a relevant volume (aportion or an entire volume) of a sample, it is essential to be precise,uniform and easy-to-use.

One aspect of the present invention is to improve the precision,uniformity, or easy-to-use of the methods and/or devices that regulate athickness of a relevant volume of a sample by compressing the samplewith two plates.

9.1 A Method for Uniformly Binding an Entity in a Sample into a BindingSite of a Plate

UAB1. A method for uniformly binding an entity in a sample into abinding site of a plate, comprising:

-   -   (a) obtaining a sample that contains target entity which are        capable of diffusing in the sample;    -   (b) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        the first plate has, on its surface, a binding site that is        configured to bind the target entity, wherein one or both of the        plates comprise spacers and each of the spacers is fixed with        its respective plate and has a predetermined height;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are either partially or completely separated apart and        the spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and the relevant        volume of the sample are between the plates, the binding site is        in contact with the relevant volume, the thickness of the        relevant volume of the sample is regulated by the plates and the        spacers and is, compared to the plates are in the open        configuration, thinner than the maximum thickness of the sample        and more uniform over the binding site;        -   wherein the spacers and the plate are configured to make the            regulated thickness of the relevant volume at the plate            closed configuration more uniform than that in the plate            open configuration; and wherein the relevant volume is a            portion or an entire volume of the sample.        -   It further has a storage site on the plate opposite to the            binding site for forming a uniform sandwich.

UAB2. A device for uniformly binding an entity in a sample into abinding site on a plate, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein the first plate has, on its surface, a binding site that        is configured to bind the target entity, wherein one or both of        the plates comprise spacers and each of the spacers is fixed        with its respective plate and has a predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the relevant volume of the        sample are between the plates, the binding site is in contact        with the relevant volume, the thickness of the relevant volume        of the sample is regulated by the plates and the spacers and is,        compared to the plates are in the open configuration, thinner        than the maximum thickness of the sample and more uniform over        the binding site;    -   wherein the spacers and the plates are configured to make the        regulated thickness of the relevant volume at the plate closed        configuration more uniform than that in the plate open        configuration; and wherein the relevant volume is a portion or        an entire volume of the sample.        9.2 A Method for Uniformly Adding a Regent on a Plate into a        Sample

UAB3. A method for uniformly adding a reagent into a relevant volume ofa sample, comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        the first plate has, on its surface, a storage site that        contains reagents to be added into a relevant volume of a        sample, the reagents are capable of being dissolving into the        sample and diffusing in the sample; and wherein one or both of        the plates comprise spacers and each of the spacers is fixed        with its respective plate and has a predetermined height;    -   (b) obtaining the sample;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and the relevant        volume of the sample are between the plates, the storage site is        in contact with the relevant volume, and the thickness of the        relevant volume of the sample is regulated by the plates and the        spacers and is thinner than the maximum thickness of the sample        when the plates are in the open configuration;        -   wherein the spacers and plates are configured to make the            thickness of the relevant volume of the sample more uniform            over the area of the relevant volume at the plate closed            configuration than that at the plate open configuration; and            wherein the relevant volume is a portion or an entire volume            of the sample.

UAB4. A device for uniformly adding a reagent into a relevant volume ofa sample, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein the first plate has, on its surface, a storage site that        contains reagents to be added into a relevant volume of a        sample, the reagents are capable of being dissolving into the        sample and diffusing in the sample; and wherein one or both of        the plates comprise spacers and each of the spacers is fixed        with its respective plate and has a predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the relevant volume of the        sample are between the plates, the storage site is in contact        with the relevant volume, and the thickness of the relevant        volume of the sample is regulated by the plates and the spacers        and is thinner than the maximum thickness of the sample when the        plates are in the open configuration;    -   wherein the spacers and plates are configured to make the        thickness of the relevant volume of the sample more uniform over        the area of the relevant volume at the plate closed        configuration than that at the plate open configuration; and        wherein the relevant volume is a portion or an entire volume of        the sample.

9.3 A Method for Uniformly Forming a Capture-Analyte-Detection Sandwich

UAB5. A method for uniformly a capture-analyte-detection sandwich on abinding site of a plate, comprising:

-   -   (a) obtaining a sample that contains a target analyte;    -   (b) obtaining capture agents and obtaining detection agents,        wherein the capture agents and the detection agents (are capable        to) bind to the target analyte to form a capture agent-target        analyte-detection agent sandwich;    -   (c) obtaining a first plate and a second plate that are movable        relative to each other into different configurations; wherein        the first plates has a binding site that has the capture agents        being immobilized on the site, and the second plate has a        storage site that stores the detection agents, which, when the        storage site is in contact with the sample, are capable to be        dissolved into the sample and diffuse in the sample; and wherein        one or both of the plates comprise spacers and each of the        spacers is fixed with its respective plate and has a        predetermined height;    -   (d) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (e) after (d), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and a relevant        volume of the sample are between the plates, the thickness of        the relevant volume of the sample is regulated by the plates and        the spacers and is thinner than the sample thickness when the        plates are in the open configuration, and the sample is in        contact with the binding site and the storage site;        -   wherein the spacers and plates are configured to make the            thickness of the relevant volume of the sample more uniform            over the area of the relevant volume at the plate closed            configuration than that at the plate open configuration; and            wherein the relevant volume is a portion or an entire volume            of the sample.

UAB6. A device for uniformly a capture-analyte-detection sandwich on abinding site of a plate, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;        -   wherein the first plates has a binding site that has capture            agents being immobilized on the site, and the capture agents            are capable of binding to a target analyte in a sample;        -   wherein the second plate has a storage site that stores the            detection agents, which, are capable of (a) when the storage            site is in contact with the sample, being dissolved into the            sample and diffuse in the sample; and (b) binding to the            target analyte and form a capture agent-target            analyte-detection agent sandwich;        -   wherein one or both of the plates comprise spacers and each            of the spacers is fixed with its respective plate and has a            predetermined height;        -   wherein one of the configurations is an open configuration,            in which: the two plates are either partially or completely            separated apart, the spacing between the plates is not            regulated by the spacers, and the sample is deposited on one            or both of the plates;        -   wherein another of the configuration is a closed            configuration, which is configured after the sample            deposition in the open configuration; and in the closed            configuration: the plates are facing each other, the spacers            and a relevant volume of the sample are between the plates,            the thickness of the relevant volume of the sample is            regulated by the plates and the spacers and is thinner than            the sample thickness when the plates are in the open            configuration, and the sample is in contact with the binding            site and the storage site;        -   wherein the spacers and plates are configured to make the            thickness of the relevant volume of the sample more uniform            over the area of the relevant volume at the plate closed            configuration than that at the plate open configuration; and            wherein the relevant volume is a portion or an entire volume            of the sample.

9.4 Uniform Regulating a Thickness of a Relevant Volume of a SampleBetween Two Plates.

UAB7. A method for regulating a thickness of a relevant volume of asample, comprising:

-   -   (a) obtaining a sample, wherein a thickness of a relevant volume        of the sample is to be regulated;    -   (b) obtaining two plates that are movable relative to each other        into different configurations, wherein one or both of the plates        comprise spacers, the spacers have a predetermined inter-spacer        distance and height, and each of the spacers is fixed with its        respective plate;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are either partially or completely separated apart and        the spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and the relevant        volume of the sample are between the plates, the thickness of        the relevant volume of the sample is regulated by the plates and        the spacers and is thinner than the maximum thickness of the        sample when the plates are in the open configuration;    -   wherein the spacers and plates are configured to make the        thickness of the relevant volume of the sample more uniform over        the area of the relevant volume at the plate closed        configuration than that at the plate open configuration; and        wherein the relevant volume is a portion or an entire volume of        the sample.

UAB8. A device for regulating a thickness of a relevant volume of asample, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein one or both of the plates comprise spacers, the spacers        have a predetermined inter-spacer distance and height, and each        of the spacers is fixed with its respective plate;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the relevant volume of the        sample are between the plates, the thickness of the relevant        volume of the sample is regulated by the plates and the spacers        and is thinner than the maximum thickness of the sample when the        plates are in the open configuration;    -   wherein the spacers and plates are configured to make the        thickness of the relevant volume of the sample more uniform over        the area of the relevant volume at the plate closed        configuration than that at the plate open configuration; and        wherein the relevant volume is a portion or an entire volume of        the sample

In the methods and the devices in the paragraphs of U1 to U8, theconfiguration of the spacers and plates that make the thickness of therelevant volume of the sample uniform has an embodiment described in thedisclosure.

Uniformity of Sample Thickness. In the methods and the devices in theparagraphs of U1 to U8, the uniformity of the thickness of the relevantvolume of the sample is such that the sample thickness at the closedconfiguration has a relative variation of at most 0.001%, at most 0.01%,at most 0.05%, at most 0.1%, at most 0.5%, at most 1%, at most 2%, atmost 5%, at most 10%, at most 20%, at most 30%, at most 50%, at most75%, at most 90%, less than 100%, or a range between any two of thesevalues.

In a preferred embodiment of the methods and the devices in theparagraphs of U1 to U8, the uniformity of the thickness of the relevantvolume of the sample is such that the sample thickness at the closedconfiguration has a relative variation of at most 0.1%, at most 0.5%, atmost 1%, at most 2%, at most 5%, at most 10%, at most 20%, at most 30%,at most 50%, or a range between any two of these values.

Another parameter that can be important to reduce the saturationincubation time is the uniformity of the sample thickness. If thethickness has a large variation over the binding site, the saturationincubation time can vary from location to location in the binding site,forcing a longer saturation incubation time to ensure all locations inthe binding site having reached the saturation.

10 Amplification Surface

One of current major obstacles for PoC diagnostics and for any assayswhich use a small sample volume is poor sensitivities. It is desirableto enhance the signal of an assay. One aspect of the present inventionis related to the devices and methods that put the binding site on asignal amplification surface (SAS) to amplify the signal for achievinghigher sensitivity. Signal amplification surfaces may also be referredto as signal amplification layers (SAL).

The general structures of SAL comprise nanoscalemetal-dielectric/semiconductor-metal structures, which amplifies localsurface electric field and gradient and light signals. The amplificationare the high at the location where there are the sharp (i.e. largecurvature) edges of a metal structure and the between a small gaps ofthe two metal structures. The highest enhancement regions are thosehaving both the sharp edges and the small gaps. Furthermore, thedimensions for all metallic and non-metallic micro/nanostructuresgenerally are less than the wavelength of the light the SAL amplifies(i.e., subwavelength).

In some embodiments, a SAL layer has as many of the metallic sharp edgesand the small gaps as possible. This requires having a dense group ofmetallic nanostructures with small gaps between the nanostructures. SALstructures may include several different layers. Furthermore, the SALlayer itself can be further improved by a process that can further coverthe portions of the metallic materials that do not have sharp edges andsmall gaps, as described in U.S. provisional application Ser. No.61/801,424, filed on Mar. 15, 2013, and PCT application WO2014197096,filed on Mar. 15, 2014, which are incorporated by reference for allpurposes, as well as PCT/US2014/028417 (Chou et al, “Analyte DetectionEnhancement By Targeted Immobilization, Surface Amplification, AndPixelated Reading And Analysis”), which is incorporated by referenceherein for all purposes.

One particular embodiment of a signal amplification surface is the D2PAarray (disk-coupled dots-on-pillar antenna arrays), which may alsocomprise a molecular adhesion layer that covers at least a part of saidmetallic dot structure, said metal disc, and/or said metallic back planeand, optionally, a capture agent that specifically binds to an analyte,wherein said capture agent is linked to the molecular adhesion layer ofthe D2PA array. The nanosensor can amplify a light signal from ananalyte, when said analyte is bound to the capture agent. In someembodiments, the dimension of one, several or all critical metallic anddielectric components of SAL are less than the wavelength of the lightin sensing. Details of the physical structure of disk-coupleddots-on-pillar antenna arrays, methods for their fabrication, methodsfor linking capture agents to disk-coupled dots-on-pillar antenna arraysand methods of using disk-coupled dots-on-pillar antenna arrays todetect analytes are described in a variety of publications includingWO2012024006, WO2013154770, Li et al (Optics Express 2011 19,3925-3936), Zhang et al (Nanotechnology 2012 23: 225-301); and Zhou etal (Anal. Chem. 2012 84: 4489) which are incorporated by reference forall purposes.

10.1 Amplifying Signal of Assaying a Target Entity in a Relevant Volumeof a Sample

M1. A method for amplifying the signal of assaying a target entity in arelevant volume of a sample, comprising:

-   -   (a) obtaining a sample that contains a target entity;    -   (b) obtaining two plates that are movable relative to each other        into different configurations, wherein one of the plates        comprises, on its surface, one binding site that comprises a        signal amplification surface that is configured to bind the        target entity and to amplify an optical signal which is on or        near the signal amplification surface; and wherein one or both        of the plates comprise spacers and each of the spacers is on its        respective plate and has a predetermined height;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are separated apart and the spacing between the plates is        not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and the relevant        volume of the sample are between the plates, the thickness of        the relevant volume of the sample is regulated by the plates and        the spacers and is thinner than that when the plates are in the        open configuration, and the relevant volume of the sample is in        contact with the binding site; and    -   (e) after (e), incubating, while the plates are in the closed        configuration, for a time period to allow the target entity in        the relevant volume of the sample to bind to the binding site;        -   wherein the relevant volume is a portion of the sample that            contact to the binding site when the plates are in the            closed configuration.

M2. A device for amplifying the signal in assaying a target entity in arelevant volume of a sample, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations,    -   wherein the first plate comprises, on its surface, one binding        site, and the binding site comprises a signal amplification        surface that is configured to (i) bind a target entity in a        sample and (ii) amplify an optical signal which is on or near        the signal amplification surface;    -   wherein one or both of the plates comprise spacers and each of        the spacers is on its respective plate and has a predetermined        height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates,    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the relevant volume of the        sample are between the plates, the thickness of the relevant        volume of the sample is regulated by the plates and the spacers        and is thinner than that when the plates are in the open        configuration;    -   wherein the relevant volume is a portion of the sample that        contact to the binding site when the plates are in the closed        configuration.

In some embodiments, the signal amplification surface includes at leastone of a metal-dielectric nanostructure, a metal-semiconductornanostructure, and a disk-coupled dots-on-pillar antenna array.

In some embodiments, the signal amplification surface includes a metallayer.

11 Saving Reagent Volume in Assaying in Fast Binding (S)

In the situation for binding entity in a reagent to a binding site on asurface (e.g. coating a plate with capture agent or stain a bio samplesurface), it is desirable to have a short incubation time. One approachfor a short incubation time is to increases the entity concentration ina reagent significantly. However, such approach is wasteful of theentity and hence costly, since in a short incubation time, only smallportion of the entity in the reagent that are near the binding site canreach the binding site for binding, and the rest are too far away todiffuse to the binding site for binding and are useless and wasted. Fora typical diffusion constant of common reagents in a common solutions,the typical diffusion length is about 10 um, 33 um, and 100 um,respectively, for an incubation time of 1 s (second), 10 s and 100 s. Atypical thickness of a liquid drop on a typical surface is 2.5 mm, whichis at least 25 time thicker than the above diffusion lengths, leadingsignificant waste (costly) if the incubation time is 100 s or less. Oneaspect of the present invention is to spread a drop(s) of reagent into alarge area but very thin thickness (thinner than a natural dropping) tosave the reagents and hence reduce the cost.

11-1 A Method for Saving Reagent that Contains Target Entity in Reagentsthat Bind to a Surface Binding Site by Spreading the Reagent. (theVolume has a Natural Contacting Area Less than the Binding Site)

S1. A method for saving a reagent that contains target entity that bindto a surface binding site, comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        the first plate has, on its surface, a binding site, and wherein        one or both of the plates comprise spacers and each of the        spacers is fixed with its respective plate and has a        predetermined height;    -   (b) obtaining a reagent that (i) contains target entity capable        to bind the binding site, and (ii) has a volume and a wetting        property such that the contact area of the reagent deposited on        the binding site, without contacting the other plate, is less        than the area of the binding site;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein,        in the open configuration: the two plates are partially or        completely separated apart, and the spacing between the plates        is not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration; wherein, in the closed configuration:        the plates are facing each other, the spacers and the sample are        between the plates, the sample contacts more area of the binding        site than that when the plates are in the open configuration,        and the thickness of the sample on the binding site is regulated        by the plates and the spacers, and is thinner than that when the        plates are in the open configuration.

In the method of Paragraph S1, it further comprised a step that after(d) and while the plates are in the closed configuration, incubating fora time and stopping the incubation, wherein the incubation time isapproximately equal to the time for the target entity diffusing acrossthe maximum sample thickness when the plates are in the closedconfiguration, and wherein the incubation is a process to allow theentity to bind to the binding site.

S2. A device for saving a reagent that contain target entity that bindto a surface binding site, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations,    -   wherein the first plate has, on its surface, a binding site that        binds target entity in a reagent, and wherein the binding site        has an area larger than the contact area of the reagent if the        reagent is deposited on only one of the plates, without        contacting the other plate;    -   wherein one or both of the plates comprise spacers and each of        the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the reagent is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the reagent deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the reagent are between the        plates, the reagent contacts more area of the binding site than        that when the plates are in the open configuration, and the        thickness of the reagent on the binding site is regulated by the        plates and the spacers, and is thinner than that when the plates        are in the open configuration.

12 Detection and/or Quantification of Volume and/or Concentration (Q)

Quantification and/or control of a relevant volume of a sample is usefulfor quantification and/or control of the concentration of chemicalcompounds (including analytes, entity, reagents, etc.) in the sample.

Common methods for a sample volume quantification include a use of ametered pipette (e.g., Eppendorf's “Research plus pipette, adjustable,0.5-10 pL”, SKU #3120000020), or a geometry. For PoC (point of care) orhome uses, such metering devices are inconvenient to use and/orexpensive. There are needs for simpler and cheaper methods and devicesfor the quantification and/or control of the sample volume and/or theconcentration.

One aspect of the present invention is related to the methods, devices,and systems that quantify and/or control a relevant volume of a samplethat deposited on a plate, without using a metered pipette and/or afixed microfluidic channel. The relevant volume, which can be a portionor the entire volume of the sample, is relevant to the quantificationand/or control of the concentration of target analyte and/or entity inthe sample. The methods, devices and systems in the present inventionare easy to use and low cost.

12.1 A Method for Quantifying a Relevant Volume of a Sample

Q1. A method for quantifying a relevant volume of a sample, comprising:

-   -   (a) obtaining a sample, wherein a relevant volume of the sample        is to be quantified;    -   (b) obtaining two plates that are movable relative to each other        into different configurations, wherein one or both of the plates        comprise spacers and the spacers have a predetermined        inter-spacer distance and height, and each of the spacers is        fixed with its respective plate;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are either partially or completely separated apart and        the spacing between the plates is not regulated by the spacers;    -   (d) after (c), spread the sample by bringing the plates into a        closed configuration, wherein, in the closed configuration: the        plates are facing each other, the spacers and the relevant        volume of the sample are between the plates, the thickness of        the relevant volume of the sample is regulated by the plates and        the spacers and is thinner than the maximum thickness of the        sample when the plates are in the open configuration, and at        least one of the spacers is inside the sample;    -   (e) quantifying the relevant volume of the sample while the        plates are in the closed configuration;        -   wherein the relevant volume is at least a portion of an            entire volume of the sample.

Q2. In some embodiments, a method for quantifying a relevant volume in asample, comprises:

-   -   (a) obtaining a first plate and a second plate;    -   (b) making a sample to quantified between the two plates;    -   (c) deforming the shape of the sample by compressing the two        plate that reduces the sample thickness and spreading the sample        between the plates laterally; and    -   (d) quantifying the relevant volume of the sample while the        plates are in the closed configuration;    -   wherein the relevant volume is at least a portion of an entire        volume of the sample.

12.2 A Plate for Use in Quantifying a Relevant Volume in a Sample

Q3. A plate for use in quantifying a relevant volume in a sample,comprising:

-   -   a plate that comprises, on its surface, (i) spacers that have a        predetermined inter-spacer distance and height and are fixed on        the surface, and (ii) a sample contact area for contacting a        sample with a relevant volume to be quantified,    -   wherein at least one of the spacers is inside the sample contact        area.

12.3 A Device for Use in Quantifying a Relevant Volume in a Sample

Q4. A device for quantifying a relevant volume in a sample, comprising:

-   -   a first plate and a second plate that (a) are movable relative        to each other into different configurations and (b) each has a        sample contact area for contacting a sample with a relevant        volume to be quantified,    -   wherein one or both of the plates comprise, on its surface(s),        spacers that have a predetermined inter-spacer distance and        height, and the spacers are fixed with respective plates;    -   wherein one of the configurations is an open configuration, in        which: the two plates are separated apart, the spacing between        the plates is not regulated by the spacers, and the sample is        deposited on one or both of the plates,    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the relevant volume of the        sample are between the plates, the thickness of the relevant        volume of the sample is regulated by the plates and the spacers        and is thinner than that when the plates are in the open        configuration, and at least one of the spacers is inside the        sample; and    -   wherein the relevant volume of the sample is quantified in the        closed configuration, and the relevant volume is at least a        portion of an entire volume of the sample.

12-5. Measuring a Relevant Volume of a Sample

MS1. In the present invention, the quantifying of a relevant volume ofthe sample while the plates are at a closed configuration includes, butnot limited to, each of the following five embodiments:

-   -   (a) measuring the relevant volume of the sample by a method of        mechanical, optical, electrical, or any combination of thereof;    -   (b) measuring one or several parameter(s) related to the        relevant volume of the sample independently using a method        selected from a method that is mechanical, optical, electrical,        or any combination of thereof;    -   (c) using predetermined one or several parameter(s) related to        the relevant volume of the sample (i.e. the parameter(s) of the        sample determined prior to the plates are at the closed        configuration);    -   (d) determining the relevant volume of the sample by (i)        measuring one or several parameters related to the revel vent        volume when the plates are at a closed configuration and (ii)        predetermining other parameters related to the relevant volume        before the plates are at the closed configuration;    -   (e) determining none-sample volume    -   (f) any combinations of the above (i.e. a, b and c).

In the method of paragraph MS1, the mechanical methods include, but notlimited to, a use of the spacers (i.e. the mechanical device thatregulate the spacing between the inner surfaces of the substrate and thecover-plate to a predetermined value), mechanical probe or rulers, soundwaves (e.g. reflection and/or interference of ultrasound wave to measurethe spacing), or any combination of thereof.

In the method of paragraph MS1, the optical methods include, but notlimited to, a use of light interference, or optical imaging (e.g. takinga 2D (two-dimensional)/3D (three-dimensional) image of the sample,optical imaging of multiple times (with different viewing angles,different wavelength, different phase, and/or different polarization),image processing, or any combination of thereof.

The electrical methods include, but not limited to, capacitive, orresistive or impedance measurements, or any combination of thereof.

In the method of paragraph MS1, in some embodiments, the measurement ofthe sample thickness is to measure the spacing between the innersurfaces of the two plate.

In the method of paragraph MS1, in some embodiments, the use ofpredetermined one or several parameter(s) related to the relevant volumeof the sample, wherein the predetermined parameter is the predeterminedsample thickness that is regulated by the spacers when the plates are ina closed configuration.

In the method of paragraph MS1, in some embodiments, the use ofpredetermined one or several parameter(s) related to the relevant volumeof the sample, wherein the predetermined parameter is the predeterminedthe spacer height.

In the method of paragraph of MS1, in some embodiments, the parametersrelated to the relevant volume of the sample are the parameters at aclosed configuration, that include, but not limited to, (i) the spacingbetween the inner surfaces of the first plate and the second plate (inCROF), (ii) the sample thickness, (iii) the entire or a relevant portionof the sample area, (iv) the entire or a relevant portion of the samplevolume, or (v) any combination of thereof.

In the method of paragraph MS1, in some embodiments, the quantificationof the sample volume or a relevant sample volume, comprising steps of(i) multiplying the sample thickness by the entire sample area to getthe entire sample volume, (ii) multiplying the sample thickness by therelevant sample area to get the relevant sample volume, or (iii)multiplying the relevant sample thickness by the entire or relevantsample area to get the relevant sample volume.

In the method of paragraph MS1, in some embodiments, the measurement isto take 3D (three-dimensional) image of the relevant volume.

In the method of paragraph MS1, in some embodiments, the quantificationof the relevant volume of the sample by measuring the lateral area ofthe relevant volume of the sample, then using it with the thickness ofthe relevant volume to determine the volume of the relevant volume ofthe sample, wherein the thickness of the relevant volume is determinedfrom the information of the spacer, and the information of the spacerinclude the spacer height;

In the method of paragraph MS1, in some embodiments, the quantificationof the relevant volume of the sample by measuring the lateral area ofthe relevant volume of the sample and the spacer together, then using itwith the thickness of the relevant volume and the volume of the spacersto determine the volume of the relevant volume of the sample, whereinthe thickness of the relevant volume is determined from the inform ofthe spacer;

In the method of paragraph MS1, in some embodiments, the quantificationof the relevant volume of the sample by measuring the lateral area andthe thickness of the relevant volume of the sample;

In the method of paragraph MS1, in some embodiments, the quantificationof the relevant volume of the sample by measuring the volume of therelevant volume of the sample optically.

In the method of paragraph MS1, in some embodiments, scale marks areused to assist the quantification of a relevant volume of the samplewhile the plates are at a closed configuration, wherein some embodimentsof the scale markers, their use and measurements, etc. are described inSection 2.

In the method of paragraph MS1, in some embodiments, the quantificationof the relevant volume of the sample comprises a step of subtracting thenone-sample volume, wherein the none-sample volume is determined, insome embodiments, by the embodiments described in in the disclosures

12-4. A Method for Quantifying Analytes Concentration in a RelevantVolume of a Sample

Q5. A method for quantifying analytes in a relevant volume of a sample,comprising:

-   -   (a) perform the steps in the method of paragraph Q1; and    -   (b) measuring, after step (a), a signal related to the analytes        from the relevant volume,        -   wherein the relevant volume is at least a portion of an            entire volume of the sample.

Q6. A method for quantifying analytes in a relevant volume of a sample,comprising:

-   -   (a) perform the steps in the method of paragraph Q2; and    -   (b) measuring, after step (a), a signal related to the analytes        from the relevant volume,        -   wherein the relevant volume is at least a portion of an            entire volume of the sample.

In the method of any of paragraphs Q5-6, in some embodiments, it furthercomprises a step of calculating the analytes concentration by dividingthe signal related to the analytes from the relevant volume of thesample by the volume of the relevant volume.

In the method of any of paragraphs Q5-6, one or both plates furthercomprise a binding site, a storage site, or both.

In the method of any of paragraphs Q5-6, in some embodiments, the signalrelated to the analyte is a signal directly from the analytes or a labelattached to the analyte.

Q7. A method for quantifying analytes in a relevant volume of a sample,comprising:

-   -   (a) perform the steps in the method of paragraph Q1, wherein one        or both plates further comprise a binding site; and    -   (b) measuring, after step (a), a signal related to the analytes        from the relevant volume,    -   wherein the relevant volume is at least a portion of an entire        volume of the sample.

Q8. A method for quantifying analytes in a relevant volume of a sample,comprising:

-   -   (a) perform the steps in the method of paragraph Q2, wherein one        or both plates further comprise a binding site; and    -   (b) measuring, after step (a), a signal related to the analytes        from the relevant volume,    -   wherein the relevant volume is at least a portion of an entire        volume of the sample.

In the method of any of paragraphs Q7-8, in some embodiments, the signalrelated to the analyte is a signal directly from the analytes that bindsto the binding site or a label attached to the analyte that binds to thebinding site.

12.5 A Plate for Use in Quantifying Analyte Concentration in a RelevantVolume in a Sample

Q9. A plate for use in quantifying analyte concentration in a relevantvolume in a sample, comprising:

-   -   a plate that comprises, on its surface, (i) spacers that have a        predetermined inter-spacer distance and height, and (ii) a        sample contact area for contacting a sample with analyte        concentration in a relevant volume to be quantified,    -   wherein at least one of the spacers is inside the sample contact        area.

12.6 A Device for Use in Quantifying Analyte Concentration in a RelevantVolume in a Sample

The concentration of target analytes and/or entity in a sample can bequantified or controlled, if the number of target analytes and/or entityin the sample are quantified, as well as the relevant volume of thesample is quantified.

Q10. A device for quantifying analyte concentration in a relevant volumein a sample, comprising:

a first plate and a second plate that (a) are movable relative to eachother into different configurations and (b) each has a sample contactarea for contacting a sample with analyte concentration in a relevantvolume to be quantified, wherein one or both of the plates comprise, onits surface(s), spacers that have a predetermined inter-spacer distanceand height, and each of the spacers are fixed with respective plates;

wherein one of the configurations is an open configuration, in which:the two plates are separated apart, the spacing between the plates isnot regulated by the spacers, and the sample is deposited on one or bothof the plates,

wherein another of the configuration is a closed configuration, which isconfigured after the sample deposition in the open configuration; and inthe closed configuration: the plates are facing each other, the spacersand the relevant volume of the sample are between the plates, thethickness of the relevant volume of the sample is regulated by theplates and the spacers and is thinner than that when the plates are inthe open configuration, and at least one of the spacers is inside thesample; and

wherein analyte concentration in the relevant volume of the sample isquantified in the closed configuration, and the relevant volume is atleast a portion of an entire volume of the sample.

In the device of any of paragraphs Q9 and Q10, the plate furthercomprises a binding site, or a storage site, or both. One embodiment ofthe binding site is a binding site that bind the analytes in the sample.

In the device of any of paragraphs Q9 and Q10, the plate furthercomprises a or a plurality of scale-markers, wherein some embodiments ofthe scale-markers described in Section 2.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, the measuring device includes at least one of an imager anda camera.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, the measuring device is configured to image the lateralarea of the relevant volume of the sample.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, the measuring device includes a light source to illuminatethe lateral area of the relevant volume of the sample.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, the step of calculating the concentration is to divide thetotal target analytes or the entity by the relevant sample volume.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, measuring signal is to use an optical imager to count thenumber of target analytes or entity. For example, the measurement can bea use of optical microscope to measure blood cells (red cell, whitecells, platelets) in a blood sample.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, measuring the number of target analytes or entity in asample can be an embodiment of surface-immobilization assay that catchthe target analytes or the entity on the surface.

In some embodiments, an apparatus for quantifying a volume of a sampleor detecting/quantifying an analyte in a sample comprises any of thedevices in paragraphs Q1-10, plus (1) optical imagers, and/or (2) alight source and optical imagers, etc. The optical imager includes aphotosensor, optical lenses, filters, polarizers, waveplates, beamsplitters, mechanical mounts, or any combination of thereof.

In some embodiments, the measuring of the relevant sample area or volumecomprises (i) having a marker on the first plate, the cover plate,between them, or any combination of thereof, (ii) taking optical imaging(e.g. taking a 2D (two-dimensional)/3D (three-dimensional) image of thesample and the image taking can be multiple times with different viewingangles, different wavelength, different phase, and/or differentpolarization) and (iii) image processing based on the maker and thesample images. The relevant means to be related to the determination oftarget analyte concentration.

Scanning. In some embodiments, the reading of a signal from a sampleuses a scanning method, where a reader (e.g. photodetectors or camera)reads a portion of the sample (or plate) and then moves to anotherportion of the sample (or plate), and such process continues untilcertain pre-specified port of the sample (or plate) being read. The scanreading of a sample covers all part of the sample (or the plate) or afraction of the sample (or the plate). In some embodiments, the scanreading are assisted by the location markers that indicate a location ofthe sample (or the plate). One example of the location markers is theperiodic spacers, which has a fixed period and location, or the markersfor the relevant area which also has predetermined location and size forindicating a location of the sample or plate.

13 Detection and Quantification of Analytes and Others (D)

In certain embodiments, an analyte is detected and/or quantified (i.e.assayed) by measuring a signal related to the analyte, wherein thesignal is an optical signal, electrical signal, mechanical signal,chemi-physical signal, or any combination of thereof. In someembodiments, the analyte assaying are performed when the two plates in aCROF device are close to each other. In some embodiments, the analyteassaying are performed when the two plates in a CROF device areseparated from each other.

The optical signal includes, but not limited to, light reflection,scattering, transmission, absorption, spectrum, color, emission,intensity, wavelength, location, polarization, luminescence,fluorescence, electroluminescence, chemoluminescence,electrochemiluminescence, or any combination of thereof. The opticalsignal is in the form of optical image (i.e. light signal vs location ofthe sample or device) or a lump sum of all photons coming from a givenarea or volume. A preferred wavelength of the light is in a range of 400nm to 1100 nm, a range of 50 nm to 400 nm, a range of 1 nm to 50 nm, ora range of 1100 to 30,000 nm. Another preferred wavelength is interahertz.

The electrical signal includes, but not limited to, charge, current,impedance, capacitance, resistance, or any combination of thereof. Themechanical signal includes, but not limited to, mechanical wave, soundwave, shock wave, or vibration. The chemi-physical signal includes, butnot limited to, PH value, ions, heat, gas bubbles, color change, thatare generated in an reaction.

For example, the label is a bead and the label is attached to the labelthrough an analyte specific binding process (e.g. use detection agent tobind the bead to the analyte, use capture agent to capture the analytewith bead, use a capture agent to bind the analyte and then usedetection agent to attach the bead, or other approaches. Note thecapture and detection agents bind the analyte specifically), then ameasurement is used to identify each of the beads that are attached tothe analytes, and count them.

In some embodiments, each of the analyte or the beads are sensed andcounted by optical means (such as (i) optical labels and reading of thelabels, (ii) surface plasmon resonance, (iii) optical interferences,(iv) electrical methods (e.g. capacitance, resistance, impedance, etc.),or others. The sensors can be on the surface of the first plate and/orthe second plate.

Certain embodiments may include determining the analyte concentration in(a) surface immobilization assay, (b) bulk assay (e.g., blood cellcounting), and (c) others. In some embodiments, the methods of thesample volume, the relevant volume of the sample, or the concentrationuses a smart-phone.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, the measuring a signal is to measure the number of theanalytes in the sample, or measure the number of a label being attachedto the analytes in the sample. In another embodiment of paragraph Q5,the “measuring signal” is to (a) identify each of the analyte or thelabel attached to each analyte, and (b) count their number.

In some embodiments, the analytes detection is an electrical method whenelectrodes are put on one or both of the first and second plates (thisapplies to any of the methods and devices that uses CROF). Theelectrodes measure the charge, current, capacitance, impedance, orresistance of a sample, or any combination of thereof. The electrodesmeasure an electrolyte in a sample. The electrodes have a thicknessequal or less than the thickness spacer. In some embodiments, theelectrode serve as a part of the spacers. The electrodes are made ofvarious conducting materials. A preferred electrode material is gold,silver, aluminum, copper, platinum, carbon nanotubes, or any combinationof thereof.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, the measuring uses the devices that is a camera orphotodetector plus an optional processor configured to make themeasurement.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, the concentration determining devices comprises a processorconfigured to determine the concentration from the measurements (volume,area, thickness, number of analytes, intensity)

In the method or the device of any of paragraphs of Q1-10, in someembodiments, it further comprising a concentration determining device isconfigured to determine the concentration of the target analytes in therelevant volume from the measured lateral area, the thickness, and themeasured amount of the target molecules.

More on Signal Detection Using Pixelated Reading and Analysis

In present invention, in some embodiments, the signals from the sample,analytes, and entity, binding sites, reagents, CROF plates, or anycombinations of thereof are detected and analytes. Some embodiments ofthe signal detection using pixelated reading and analysis are describedin the disclosure, while some other embodiments are described inPublication Number: WO2014144133 A and Application Number:PCT/US2014/028417 (Chou et al, “Analyte Detection Enhancement ByTargeted Immobilization, Surface Amplification, And Pixelated ReadingAnd Analysis”), which is incorporated by reference herein for allpurposes.

In some embodiments, the signal is electromagnetic signal, includingelectrical and optical signals with different frequencies, lightintensity, fluorescence, chromaticity, luminescence (electrical andchemo-luminescence), Raman scattering, time resolved signal (includingblinking). The signals also can be the forces due to local electrical,local mechanical, local biological, or local optical interaction betweenthe plate and the reading device. The signal also includes the spatial(i.e. position), temporal and spectral distribution of the signal. Thedetection signal also can be absorption.

The analyte include proteins, peptides, DNA, RNA, nucleic acid, smallmolecules, cells, nanoparticles with different shapes. The targetedanalyte can be either in a solution or in air or gas phase. The sensingincludes the detection of the existence, quantification of theconcentration, and determination of the states of the targeted analyte.

In some embodiments, electric field is used to assist molecularselectivity, or bonding, and detection.

Detection/Reading Methods

In some embodiments of optical detection (i.e. detection byelectromagnetic radiation), the methods include, but not limited to,far-field optical methods, near-field optical methods, epi-fluorescencespectroscopy, confocal microscopy, two-photon microscopy, and totalinternal reflection microscopy, where the target analytes are labelledwith an electromagnetic radiation emitter, and the signal in thesemicroscopies can be amplified by the amplification surface of a CROFplate.

In some embodiments, the signal comprises the information of theposition, local intensity, local spectrum, local polarization, localphase, local Raman signature of said signals, or any combination ofthereof.

In some embodiments, the detection of a signal is to measure a lump-sumsignal from an area (i.e. the signal from the area, regardless whichlocation in the area).

In certain embodiments, the detection of signal is to measure an signalimage of an area (i.e. signal vs location); namely, the area is dividedinto pixels and the signal from each pixel of the area is individuallymeasured, which is also termed “PIX” or “pixelated imaging detection”.The individual measurement of each pixel can be in parallel orsequential or a mix.

In some embodiments, the reading uses appropriate detecting systems forthe signal to be detected in sequence or in parallel or theircombination. In a sequential detection, one or several pixels aredetected a time, and scanner will be used to move the detection intoother areas of the SAL. In a parallel detection, a multipixel detectorarray, such as imaging camera (e.g. CCD's), will be used to take detectthe signals from different pixels at the same time. The scan can besingle path or multi-path with a different pixel size for each path.FIG. 2C of PCT/US2014/028417 schematically illustrates pixelated readingon an x, y, z stage.

The pixel size for the reading/detection will be adjusted to for thebalance of optical resolution and total reading time. A smaller pixelsize will take a longer time for reading/scanning the entire or fractionof the SAL. A typical pixel size is 1 um to 10 um in size. The pixel hasdifferent shapes: round, square and rectangle. The lower limit of thepixel size is determined by the optical resolution of the microscopesystem, and the higher limit of the pixel size is determined in order toavoid reading error from the uneven optical response of the imager(optical aberration, illumination uniformity, etc.).

Reading System

Referred to the Figures in of PCT/US2014/028417, an embodiment of areading system comprises (a) a plate or plates used for CROF; (b) areading device 205 for producing an image of signals emanating from asurface of said plate, wherein signals represent individual targetedanalyte binding events; (c) a device assembly 300 that holds the plateand the imager; (d) an electronics and a data storage 301 for storingsaid image; and (e) a computer comprising programming for identifyingand counting individual binding events in an area of the image.

The device assembly 300 controls or changes the relative positionbetween the plate and the reading device, in at least one of the three(x, y, z) orthogonal directions, for reading the signal. An embodimentof the device assembly comprises a scanner 301. In some embodiments, thescanner 301 scans in in at least one of the three (x, y, z) orthogonaldirections.

In some embodiments, the reading device 302 is a CCD camera. In someembodiments, the reading device 302 is a photodetector comprising one ormore other optical devices that are selected from optical filters 303,spectrometer, lenses 304, apertures, beam splitter 305, mirrors 306,polarizers 307, waveplates, and shutters. In some embodiments, hereading device 302 is a smartphone or mobile phone, which have thecapability of local and remote communications. The reading devicecollects the position, local intensity, local spectrum, local Ramansignature of said signals, or any combination of thereof.

In some embodiments, optical filters 303, light beam splitters 305,optical fibers, a photodetector (e.g. pn junction, a diode, PMT(photomultiplier tube), or APD (Avalanch Photo Diode), imaging camera(e.g. CCD's, or cellphone camera) and spectrometer together with ascanner provided by the device assembly 301 are coupled to a microscopesystem which uses a far-field confocal setting or a wide-field viewsetting.

In some embodiments, in confocal setting, the reading is performed byrecording the brightness, temporal change and spectral change of one ora few pixels a time and raster scanning the entire interested area ofthe SAL. In some embodiments, in wide-field view setting, a camera isused to record the brightness and temporal change of the entire or afraction of SAL area a time. In some embodiments, proper optical filtersand light beam manipulators (polarizer, beam splitters, optical fibers,etc.) is need to ensure only the desired signal is collected anddetected. FIG. 9 of PCT/US2014/028417 schematically illustrates onearrangement of components for this system. In some embodiments, theanalysis comprises of an imaging processing methods, including, notlimited to, the methods in Open-CV or Image-J.Pixelated Analysis (PIX). In some embodiments of PIX, the signalsdetected in a pixelated manner are analyzed to determine the numberand/or types of the particular molecules at a particular pixel orseveral pixels, which, in turn is used to quantify the type and/orconcentration of the targeted analytes. The term “signal detected in apixelated manner” refers to the method where the area that has signal(s)is divided into pixels and the signal from each pixel of the area isindividually measured, which is also termed “PIX” or “pixelated imagingdetection”. The individual measurement of each pixel can be in parallelor sequential or a mix.

In some embodiments, the analysis comprises to analyze the spatial,tempo, spectral information of the signal. In some embodiments, theanalysis include, but not limited to, statistical analysis, comparison,integration, and others. FIG. 5 of PCT/US2014/028417 shows a flow chartfor one embodiment of this method.

14 Labels

One or any combinations of the embodiments of the optical labelsdescribed in the entire disclosure applies to all the methods anddevices described in the entire description of the present invention.

In some embodiments, a label(s) is attached to a detection agent(s), ananalyte(s) or an entity (ties). In certain embodiments, the label is anoptical label, an electric label, enzymes that can be used to generatean optical or electrical signal, or any combination of thereof. Incertain embodiments, a detection agent(s), an analyte(s) or an entity(ties) are attached a connection molecule (e.g. protein, nucleic acid,or other compounds) which later is attached to a label. In certainembodiments, cells (e.g. blood cells, bacteria, etc.) or nanoparticlesare stained by a labels. In some embodiments, an optical label is anobject that can generate an optical signal, wherein the generation ofthe optical signal includes, but not limited to, light (i.e. photon's)reflection, scattering, transmission, absorption, spectrum, color,emission, intensity, wavelength, location, polarization, luminescence,fluorescence, electroluminescence, photoluminescence (fluorescence),chemoluminescence, electrochemiluminescence, or any combination ofthereof. In some embodiments, the optical signal is in the form ofoptical image (i.e. light signal vs location of the sample or device) ora lump sum of all photons coming from a given area or volume. Apreferred wavelength of the light is in a range of 400 nm to 1100 nm, arange of 50 nm to 400 nm, a range of 1 nm to 50 nm, or a range of 1100to 30,000 nm. Another preferred wavelength is in terahertz.

Beads, nanoparticles, and quantum dots. In some embodiments, the opticallabel is beads, nanoparticles, quantum dots, or any combination ofthereof.

In some embodiments, the diameter of the bead, nanoparticles, or quantumdots is 1 nm or less, 2 nm or less, 5 nm or less, 10 nm or less, 20 nmor less, 30 nm or less, 40 nm or less, 50 nm or less, 60 nm or less, 70nm or less, 80 nm or less, 100 nm or less, 120 nm or less, 200 nm orless, 300 nm or less, 500 nm or less, 800 nm or less, 1000 nm or less,1500 nm or less, 2000 nm or less, 3000 nm or less, 5000 nm or less, or arange between any two of the values.

In some embodiments, the beads or quantum dots are used as labels andthey are precoated on the plates of CROF and the inner spacing betweenthe two plates are 1 um or less, 10 um or less, 50 um or less, or arange between any two of the values.

In some embodiment, the separation between the beads in a solution

-   -   Diffusion time. (The thickness of the relevant volume of the        transfer medium leads to the diffusion time of an optical label        across the thickness, to be less than 1 ms,    -   The dissolving time can controlled. The control can use photon,        heat or other exsiccations and their combinations. The        dissolving will not start until an excitation energy is applied.

In some embodiments of the label are nanoparticles that has a diameterof 10 nm or larger. The nanoparticles of such large diameter has lessdiffusion constant than small molecules (mass <1000 Da) and largemolecules (mass=1,000 to 1,000,000 Dalton (da), leading to a longerdiffusion time for a given solution and distance. To reduce thediffusion time, is to reduce the diffusion distance.

They have particular advantages over the prior art, when the opticallabels are beads or other nanoparticles that have a diameter large thana few nanometers. This is because that the diffusion constant of anobject in a liquid is, for the first order approximation, inverselyproportional to the diameter of the object (according to Einstein-Stokesequation).

For example, a bead optical label with a diameter of 20 nm, 200, and2000 nm respectively has a diffusion constant and hence a diffusion time10, 100, and 1000 times larger and longer than that for a bead of 2 nm.For a typical diffusion distance used in current assays, this would leadto a long saturation incubation time that is in practical for PoC (Pointof Care) applications.

However, the present invention has solved the long incubation time foroptical labels with a diameter larger than a few nanometers. The presentinvention has the optical label stored on a plate surface, and thenplaces the storage surface next to binding site with a separate distance(between the two) in sub-millimeter, microns or even nanometer scale andfill the separation gap by a transfer medium (where the stored opticallabel dissolved into the transfer medium and diffuse to the bindingsite). The present invention also able to control such small distanceuniformly over large binding site area and easily by using spacertechnologies.

Labeling the analyte may include using, for example, a labeling agent,such as an analyte specific binding member that includes a detectablelabel. Detectable labels include, but are not limited to, fluorescentlabels, colorimetric labels, chemiluminescent labels, enzyme-linkedreagents, multicolor reagents, avidin-streptavidin associated detectionreagents, and the like. In certain embodiments, the detectable label isa fluorescent label. Fluorescent labels are labeling moieties that aredetectable by a fluorescence detector. For example, binding of afluorescent label to an analyte of interest may allow the analyte ofinterest to be detected by a fluorescence detector. Examples offluorescent labels include, but are not limited to, fluorescentmolecules that fluoresce upon contact with a reagent, fluorescentmolecules that fluoresce when irradiated with electromagnetic radiation(e.g., UV, visible light, x-rays, etc.), and the like.

In certain embodiments, suitable fluorescent molecules (fluorophores)for labeling include, but are not limited to, IRDye800CW, Alexa 790,Dylight 800, fluorescein, fluorescein isothiocyanate, succinimidylesters of carboxyfluorescein, succinimidyl esters of fluorescein,5-isomer of fluorescein dichlorotriazine, cagedcarboxyfluorescein-alanine-carboxamide, Oregon Green 488, Oregon Green514; Lucifer Yellow, acridine Orange, rhodamine, tetramethylrhodamine,Texas Red, propidium iodide, JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazoylcarbocyanineiodide), tetrabromorhodamine 123, rhodamine 6G, TMRM (tetramethylrhodamine methyl ester), TMRE (tetramethyl rhodamine ethyl ester),tetramethylrosamine, rhodamine B and 4-dimethylaminotetramethylrosamine,green fluorescent protein, blue-shifted green fluorescent protein,cyan-shifted green fluorescent protein, red-shifted green fluorescentprotein, yellow-shifted green fluorescent protein,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives, such as acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphth-alimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide;4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a diaza-5-indacene-3-propioni-cacid BODIPY; cascade blue; Brilliant Yellow; coumarin and derivatives:coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2-, 2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-(dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives: eosin, eosin isothiocyanate, erythrosin and derivatives:erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5-carboxyfluorescein(FAM),5-(4,6-dichlorotriazin-2-yl)amino-fluorescein (DTAF),2′,7′dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelli-feroneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl hodamine isothiocyanate (TRITC); riboflavin;5-(2′-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), rosolic acid; CALFluor Orange 560; terbium chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7;IRD 700; IRD 800; La Jolla Blue; phthalocyanine; and naphthalocyanine,coumarins and related dyes, xanthene dyes such as rhodols, resorufins,bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazidessuch as luminol, and isoluminol derivatives, aminophthalimides,aminonaphthalimides, aminobenzofurans, aminoquinolines,dicyanohydroquinones, fluorescent europium and terbium complexes;combinations thereof, and the like. Suitable fluorescent proteins andchromogenic proteins include, but are not limited to, a greenfluorescent protein (GFP), including, but not limited to, a GFP derivedfrom Aequorea victoria or a derivative thereof, e.g., a “humanized”derivative such as Enhanced GFP; a GFP from another species such asRenilla reniformis, Renilla mulleri, or Ptilosarcus guernyi; “humanized”recombinant GFP (hrGFP); any of a variety of fluorescent and coloredproteins from Anthozoan species; combinations thereof; and the like.

In certain embodiments, the dyes can be used to stain the blood cellscomprise Wright's stain (Eosin, methylene blue), Giemsa stain (Eosin,methylene blue, and Azure B), May-Grünwald stain, Leishman's stain(“Polychromed” methylene blue (i.e. demethylated into various azures)and eosin), Erythrosine B stain (Erythrosin B), and other fluorescencestain including but not limit to Acridine orange dye,3,3-dihexyloxacarbocyanine (DiOC6), Propidium Iodide (PI), FluoresceinIsothiocyanate (FITC) and Basic Orange 21 (BO21) dye, Ethidium Bromide,Brilliant Sulfaflavine and a Stilbene Disulfonic Acid derivative,Erythrosine B or trypan blue, Hoechst 33342, Trihydrochloride,Trihydrate, and DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride).

In certain embodiments, the labeling agent is configured to bindspecifically to the analyte of interest. In certain embodiments, alabeling agent may be present in the CROF device before the sample isapplied to the CROF device. In other embodiments, the labeling agent maybe applied to the CROF device after the sample is applied to the CROFdevice. In certain embodiments, after the sample is applied to the CROFdevice, the CROF device may be washed to remove any unbound components,e.g. un bound analyte and other non-analyte components in the sample,and the labeling agent may be applied to the CROF device after thewashing to label the bound analyte. In some embodiments, the CROF devicemay be washed after the labeling agent is bound to the analyte-captureagent complex to remove from the CROF device any excess labeling agentthat is not bound to an analyte-capture agent complex.

In certain embodiments, the analyte is labeled after the analyte isbound to the CROF device, e.g., using a labeled binding agent that canbind to the analyte simultaneously as the capture agent to which theanalyte is bound in the CROF device, i.e., in a sandwich-type assay. Insome embodiments, a nucleic acid analyte may be captured on the CROFdevice, and a labeled nucleic acid that can hybridize to the analytesimultaneously as the capture agent to which the nucleic acid analyte isbound in the CROF device.

In certain aspects, a CROF device enhances the light signal, e.g.,fluorescence or luminescence, that is produced by the detectable labelbound directly or indirectly to an analyte, which is in turn bound tothe CROF device. In certain embodiments, the signal is enhanced by aphysical process of signal amplification. In some embodiments, the lightsignal is enhanced by a nanoplasmonic effect (e.g., surface-enhancedRaman scattering). Examples of signal enhancement by nanoplasmoniceffects is described, e.g., in Li et al, Optics Express 2011 19:3925-3936 and WO2012/024006, which are incorporated herein by reference.In certain embodiments, signal enhancement is achieved without the useof biological/chemical amplification of the signal. Biological/chemicalamplification of the signal may include enzymatic amplification of thesignal (e.g., used in enzyme-linked immunosorbent assays (ELISAs)) andpolymerase chain reaction (PCR) amplification of the signal. In otherembodiments, the signal enhancement may be achieved by a physicalprocess and biological/chemical amplification.

Sensitivity. In certain embodiments, the CROF device is configured tohave a detection sensitivity of 0.1 nM or less, such as 10 pM or less,or 1 pM or less, or 100 fM or less, such as 10 fM or less, including 1fM or less, or 0.5 fM or less, or 100 aM or less, or 50 aM or less, or20 aM or less. In certain embodiments, the CROF device is configured tohave a detection sensitivity in the range of 10 aM to 0.1 nM, such as 20aM to 10 pM, 50 aM to 1 pM, including 100 aM to 100 fM. In someinstances, the CROF device is configured to be able to detect analytesat a concentration of 1 ng/mL or less, such as 100 pg/mL or less,including 10 pg/mL or less, 1 pg/mL or less, 100 fg/m L or less, 10fg/mL or less, or 5 fg/m L or less. In some instances, the CROF deviceis configured to be able to detect analytes at a concentration in therange of 1 fg/mL to 1 ng/mL, such as 5 fg/m L to 100 pg/mL, including 10fg/m L to 10 pg/mL. In certain embodiments, the CROF device isconfigured to have a dynamic range of 5 orders of magnitude or more,such as 6 orders of magnitude or more, including 7 orders of magnitudeor more.Reading. In certain instances, the period of time from applying thesample to the CROF device to reading the CROF device may range from 1second to 30 minutes, such as 10 seconds to 20 minutes, 30 seconds to 10minutes, including 1 minute to 5 minutes. In some instances, the periodof time from applying the sample to the signal enhancing detector togenerating an output that can be received by the device may be 1 hour orless, 30 minutes or less, 15 minutes or less, 10 minutes or less, 5minutes or less, 3 minutes or less, 1 minute or less, 50 seconds orless, 40 seconds or less, 30 seconds or less, 20 seconds or less, 10seconds or less, 5 seconds or less, 2 seconds or less, 1 second or less,or even shorter. In some instances, the period of time from applying thesample to the signal enhancing detector to generating an output that canbe received by the device may be 100 milliseconds or more, including 200milliseconds or more, such as 500 milliseconds or more, 1 second ormore, 10 seconds or more, 30 seconds or more, 1 minute or more, 5minutes or more, or longer.

Any suitable method may be used to read the CROF device to obtain ameasurement of the amount of analyte in the sample. In some embodiments,reading the CROF device includes obtaining an electromagnetic signalfrom the detectable label bound to the analyte in the CROF device. Incertain embodiments the electromagnetic signal is a light signal. Thelight signal obtained may include the intensity of light, the wavelengthof light, the location of the source of light, and the like. Inparticular embodiments, the light signal produced by the label has awavelength that is in the range of 300 nm to 900 nm. In certainembodiments, the light signal is read in the form of a visual image ofthe CROF device.

In certain embodiments, reading the CROF device includes providing asource of electromagnetic radiation, e.g., light source, as anexcitation source for the detectable label bound to the biomarker in theCROF device. The light source may be any suitable light source to excitethe detectable label. Exemplary light sources include, but are notlimited to, sun light, ambient light, UV lamps, fluorescent lamps,light-emitting diodes (LEDs), photodiodes, incandescent lamps, halogenlamps, and the like.

Reading the CROF device may be achieved by any suitable method tomeasure the amount of analyte that is present in the sample and bound tothe CROF device. In certain embodiments, the CROF device is read with adevice configured to acquire the light signal from the detectable labelbound to the analyte in the CROF device. In some cases, the device is ahandheld device, such as a mobile phone or a smart phone. Any suitablehandheld device configured to read the CROF device may be used in thedevices, systems and methods in the present invention. Certain deviceembodiments configured to read the CROF device are described in, e.g.,U.S. Provisional Application Ser. No. 62/066,777, filed on Oct. 21,2014, which is incorporated herein by reference.

In some embodiments, the device includes an optical recording apparatusthat is configured to acquire a light signal from the CROF device, e.g.,acquire an image of the CROF device. In certain instances, the opticalrecording apparatus is a camera, such as a digital camera. The term“digital camera” denotes any camera that includes as its main componentan image-taking apparatus provided with an image-taking lens system forforming an optical image, an image sensor for converting the opticalimage into an electrical signal, and other components, examples of suchcameras including digital still cameras, digital movie cameras, and Webcameras (i.e., cameras that are connected, either publicly or privately,to an apparatus connected to a network to permit exchange of images,including both those connected directly to a network and those connectedto a network by way of an apparatus, such as a personal computer, havingan information processing capability). In one example, reading the CROFdevice may include video imaging that may capture changes over time. Forexample, a video may be acquired to provide evaluation on dynamicchanges in the sample applied to the CROF device.

In certain embodiments, the optical recording apparatus has asensitivity that is lower than the sensitivity of a high-sensitivityoptical recording apparatus used in research/clinical laboratorysettings. In certain cases, the optical recording apparatus used in thesubject method has a sensitivity that is lower by 10 times or more, suchas 100 times or more, including 200 times or more, 500 times or more, or1,000 times or more than the sensitivity of a high-sensitivity opticalrecording apparatus used in research/clinical laboratory settings.

In certain embodiments, the device may have a video display. Videodisplays may include components upon which a display page may bedisplayed in a manner perceptible to a user, such as, for example, acomputer monitor, cathode ray tube, liquid crystal display, lightemitting diode display, touchpad or touchscreen display, and/or othermeans known in the art for emitting a visually perceptible output. Incertain embodiments, the device is equipped with a touch screen fordisplaying information, such as the image acquired from the detectorand/or a report generated from the processed data, and allowinginformation to be entered by the subject.

15 Multiplexing

In any embodiment described herein, the system may be designed forperforming a multiplex assay and, as such, may contain multiple storagesites, multiple binding sites, or multiple storage sites and multiplebinding sites such that different assays can be performed on differentareas on the surface of one of the plates. For example, in oneembodiment, in one embodiment, one of the plates may contain multiplebinding site that each contain a different capture agent, therebyallowing the detection of multiple analytes in the sample in the sameassay. The sites may be spatially separated from, although proximal to,one another.

FIG. 10 schematically illustrates an exemplary embodiment of the presentinvention, a multiplexed detection in a single CROF device using onebinding site one plate and a plurality of storage sites on the otherplate. Panel (a) and (b) is a perspective and a cross-sectional view ofan exemplary device, respectively. In the exemplary case, themultiplexed CROF device comprises a first plate and a second plate,wherein one surface of the first plate has one binding site; wherein onesurface of the second plate has a plurality of storage sites; andwherein different storage sites can have the same detection agent but ofdifferent concentrations or can have different detection agents of thesame or different concentrations. In some embodiments, the area of thebinding site is larger that of each storage site. In some embodiments,the binding site area is larger than the total area of all storagesites, and/or the binding site area is aligned with the storage sites(i.e. they are top each other, namely, the shortest distance between thebinding site and a point on the storages are the same or nearly thesame).

FIG. 11 schematically illustrates a further exemplary embodiment of thepresent invention, a multiplexed detection in a single CROF device usingone storage site on one plate and multiple binding sites on the otherplate. Panel (a) and (b) is a perspective and a cross-sectional view ofan exemplary device, respectively. In the exemplary case, themultiplexed CROF device comprises a first plate and a second plate,wherein one surface of the first plate has multiple binding sites;wherein one surface of the second plate has one storage site; andwherein different binding sites can have the same capture agent but ofdifferent concentrations or can have different capture agents of thesame or different concentrations. In some embodiments, the area of thestorage site is larger that of each storage site. In some embodiments,the storage site area is larger than the total area of all bindingsites, and/or is aligned with the binding sites (i.e. they are top eachother).

FIG. 12 schematically illustrates a further exemplary embodiment of thepresent invention, a multiplexed detection in a single CROF device withmultiple binding sites on one plate and multiple corresponding storagesites on another plate. Panel (a) and (b) is a perspective and across-sectional view of an exemplary device, respectively. In theexemplary case, a multiplexed CROF device comprises a first plate and asecond plate, wherein one surface of the first plate has a plurality ofbinding sites; wherein one surface of the second plate has a pluralityof corresponding storage sites; wherein each corresponding storage siteis located in a location on the second plate that is corresponding tothe location of a binding site on the first plate, so that when theplates are placed face-to-face, each binding site overlaps with only onestorage site and each storage site overlaps with only one storage site;wherein different storage sites can have the same detection agent but ofdifferent concentrations or can have different detection agents of thesame or different concentrations; and wherein different storage sitescan have the same capture agent but of different concentrations or canhave different capture agents of the same or different concentrations.

In certain embodiments, the device of any of FIGS. 10, 11, and 12 ,wherein the first plate further comprises, on its surface, a firstpredetermined assay site and a second predetermined assay site, whereinthe distance between the edges of the neighboring multiple assay sitesis substantially larger than the thickness of the uniform thicknesslayer when the plates are in the closed position, wherein at least apart of the uniform thickness layer of the sample is over thepredetermined assay sites, and wherein the sample has one or a pluralityof analytes that are capable of diffusing in the sample. By making thedistance between the edges of the neighboring multiple assay sites largethan the sample thickness, it makes it possible to have multiple bindingsites without fluidically isolated the different portion of a sample,since an saturation incubation of the assay can complete between asignificant inter-diffusion between the two neighboring sites. Byproperly choosing the ratio of the neighboring distance to the samplethickness and properly selecting the measurement time between a timelonger than the assay saturation incubation time but less than a timefor a significant inter-diffusion between two neighboring sites, one cando multiplexing by CROF without isolating different part of a sample. Insome embodiments, the ratio of the neighbor distance to the samplethickness at the closed configuration is 1.5 or larger, 3 or larger, 5or larger, 10 or larger, 20 or larger, 30 or larger, 50 or larger, 100or larger, 200 or larger, 1000 or larger, 10,000 or larger, or a rangebetween any two of the values. The ratio is 3 or larger for a preferredembodiment, 5 or larger for another preferred embodiment, 10 or largerfor a certain preferred embodiment, 30 or larger for another preferredembodiment, and 100 or larger for another preferred embodiment.

In certain embodiments, the device of any of FIGS. 10, 11, and 12 ,wherein the first plate has, on its surface, at least three analyteassay sites, and the distance between the edges of any two neighboringassay sites is substantially larger than the thickness of the uniformthickness layer when the plates are in the closed position, wherein atleast a part of the uniform thickness layer is over the assay sites, andwherein the sample has one or a plurality of analytes that are capableof diffusing in the sample.

In certain embodiments, the device of any of FIGS. 10, 11, and 12 ,wherein the first plate has, on its surface, at least two neighboringanalyte assay sites that are not separated by a distance that issubstantially larger than the thickness of the uniform thickness layerwhen the plates are in the closed position, wherein at least a part ofthe uniform thickness layer is over the assay sites, and wherein thesample has one or a plurality of analytes that are capable of diffusingin the sample.

The method or the devices of any of paragraph of U1-6, X-6, P1-8, W1-6,V1-4, UAB1-8, M1-2, S1-2, Q110, and H1 as well as their any combination,wherein the first and second plate further comprise the binding site(s)and the storage site, as described in FIG. 10 , FIG. 11 , or FIG. 12 formultiplexed detection.

In these embodiments the device may for parallel, multiplex, assaying ofa liquid sample without fluidic isolation (i.e., without their being aphysical barrier between the assay regions). This device may comprise afirst plate and a second plate, wherein: i. the plates are movablerelative to each other into different configurations; one or both platesare flexible; ii. one or both of the plates comprise spacers that arefixed with a respective plate; and the spacers have a predeterminedsubstantially uniform height and a predetermined constant inter-spacerdistance; iii. each of the plates has, on its respective surface, asample contact area for contacting a sample that contains a sample thatcontains one or more target analytes which is capable of diffusing inthe sample, iii. the first plate has, on its surface, one or a pluralityof binding sites that each has a predetermined area comprising a captureagent that binds and immobilizes a corresponding target analyte of thesample; and iv the second plate has, on its surface, one or a pluralityof corresponding storage sites that each has a predetermined area andcomprises a detection agent of a concentration that, upon contacting thesample, dissolves into the sample and diffuses in the sample, whereineach capture agent, target analyte and corresponding detection agent iscapable of forming a capture agent-target analyte-detection agentsandwich in a binding site of the first plate; wherein one of theconfigurations is an open configuration, in which: the two plates areeither partially or completely separated apart, the spacing between theplates is not regulated by the spacers, and the sample is deposited onone or both of the plates, and wherein another of the configurations isa closed configuration which is configured after the sample depositionin the open configuration; and in the closed configuration: i. at leastpart of the sample is compressed into a layer of uniform thickness thatis in contact with and confined by the inner surfaces of the two platesand that covers the one or a plurality of binding sites and the one or aplurality of storage sites, ii the one or a plurality of correspondingstorage sites are over the one or a plurality of binding sites, and iii.the uniform thickness of the layer is regulated by the spacers and theplates, is less than 250 um, and is substantially less than the lineardimension of the predetermined area of each storage site; and iv. thereis no fluidic isolation between the binding site and/or the storagesites, wherein the separation between the edges of the neighboringstorage sites and the separation between the edges of the neighboringbinding sites are larger than the distance that a target analyte ordetection agent can diffuse in the relevant time, and wherein there isno fluidic isolation between the binding site sites and/or the storagesites.

In some embodiments, the first plate has, on its surface, a plurality of(at least 2, at least 4 or at least 16 or more) of the binding sites.

In some embodiments, each of said plurality of binding sites binds to adifferent target analyte.

In some embodiments, the second plate has, on its surface, a plurality(at least 2, at least 4 or at least 16 or more) of the correspondingstorage sites.

In some embodiments, each of the plurality of corresponding storagesites binds to a different target analyte.

In some embodiments, the first plate has, on its surface, a plurality ofsaid binding sites and the second plate has, on its surface, a pluralityof said corresponding storage sites, wherein each binding site faces acorresponding storage site when the plates are in the closedconfiguration.

In some embodiments, the first plate has, on its surface, a plurality ofsaid binding sites and the second plate has, on its surface, a storagesite, wherein at least some of the binding sites face an area in thestorage site when the plates are in the closed configuration.

In some embodiments the first plate has, on its surface, a binding siteand the second plate has, on its surface, a plurality of storage sites,wherein at least some of the storage sites face an area in the bindingsite when the plates are in the closed configuration.

In some embodiments the first plate has, on its surface, a plurality ofbinding sites, wherein the binding sites contain different captureagents that bind and immobilize the same target analyte.

In some embodiments the first plate has, on its surface, a plurality ofbinding sites, wherein the binding sites contain the same capture agent.

In some embodiments, the capture agent is at different densities in thedifferent binding sites. These embodiments may be used to provide a wayto quantify the amount of analyte in a sample.

In some embodiments, there is a separation between two neighboringbinding sites or two neighboring storage sites, and the ratio of theseparation to the sample thickness in the closed configuration is atleast 3, e.g., at least 5, at least 10, at least 20 or at least 50.

In some embodiments, the inter-spacer distance is in the range of 1 umto 120 um.

In some embodiments, the flexible plates have a thickness in the rangeof 20 um to 250 um (e.g., in the range of 50 um to 150 um) and Young'smodulus in the range 0.1 to 5 GPa (e.g., in the range of 0.5-2 GPa).

In some embodiments, the thickness of the flexible plate times theYoung's modulus of the flexible plate is in the range 60 to 750 GPa-um.

In some embodiments, this method may comprise (a) obtaining a samplethat contains one or more target analytes, which are capable ofdiffusing in the sample; (b) obtaining a first and second plates thatare movable relative to each other into different configurations,wherein: i. one or both of the plates comprise spacers that are fixedwith a respective plate and one or both plates are flexible, ii. thespacers have a predetermined substantially uniform height and apredetermined constant inter-spacer distance, iii. the first plate has,on its surface, one or a plurality of binding sites that each has apredetermined area comprising a capture agent that binds and immobilizesa corresponding target analyte of (a); and iv. the second plate has, onits surface, one or a plurality of corresponding storage sites that eachhas a predetermined area and comprises a detection agent of aconcentration that, upon contacting the sample, dissolves into thesample and diffuses in the sample, wherein each capture agent, targetanalyte and corresponding detection agent is capable of forming acapture agent-target analyte-detection agent sandwich in a binding siteof the first plate; (c) depositing the sample on one or both of theplates when the plates are configured in an open configuration, whereinthe open configuration is a configuration in which the two plates areeither partially or completely separated apart and the spacing betweenthe plates is not regulated by the spacers; (d) after (c), compressingthe sample by bringing the two plates into a closed configuration,wherein the closed configuration is a configuration in which: i. atleast part of the sample is compressed into a layer of uniform thicknessthat is in contact with and confined by the inner surfaces of the twoplates and that is in contact with the one or a plurality of bindingsites and the one or a plurality of storage sites, ii the one or aplurality of corresponding storage sites are over the one or a pluralityof binding sites, and iii. the uniform thickness of the layer isregulated by the spacers and the plates, is less than 250 um, and issubstantially less than the linear dimension of the predetermined areaof each storage site; (e) after (d) and while the plates are in theclosed configuration, either: (1) incubating the sample for a relevanttime length and then stopping the incubation; or (2) incubating thesample for a time that is equal or longer than the minimum of a relevanttime length and then assessing, within a time period that is equal orless than the maximum of the relevant length of time, the binding ofeach target analyte to a binding site; wherein the relevant time lengthis: i. equal to or longer than the time that it takes for a targetanalyte of (a) to diffuse across the thickness of the uniform thicknesslayer at the closed configuration; and ii. significantly shorter thanthe time that it takes a target analyte of (a) to laterally diffuseacross the smallest linear dimension of the predetermined area of astorage site or binding site; thereby producing a reaction in which, atthe end of the incubation in (1) or during the assessing in (2), themajority of the capture agent-target analyte-detection agent sandwichbound to each binding site is from a corresponding relevant volume ofthe sample; wherein the incubation allows each target analyte to bind toa binding site and a detection agent, wherein the corresponding relevantvolume is a portion of the sample that is above the correspondingstorage site at the closed configuration, wherein the separation betweenthe edges of the neighboring storage sites and the separation betweenthe edges of the neighboring binding sites are larger than the distancethat a target analyte or detection agent can diffuse in the relevanttime, and wherein there is no fluidic isolation between the binding sitesites and/or the storage sites.

Any embodiment of the multiplex assay device described above may be usedin this method.

16 Small Volume Samples or Reagent in Wide Well (E)

In some applications, a well on a plate will be used for testing asample with a sample volume small relative to the area of the well thatsample must cover. One aspect of the present invention is the methodsand devices that allow assaying and other chemical reactions of a smallvolume of sample or reagent in a wide well. The term “well” refers to ahollow compartment, recessed area, or a depression on a surface, whichprevents a liquid deposited inside the well from flowing outside of thewell by the well solid bottom and the enclosed sidewall (FIG. 8 ). Thearea of the well is the area enclosed by the sidewall. The term “a smallvolume of a sample” and “a wide well” mean that when the sample isdropped onto the well bottom and without any device to spread thesample, the volume of the sample on the well bottom has a contact areawith the well bottom is less than the well area (i.e. the small and wideis a comparison of the sample natural contact area and the well bottomarea). The well plays a role of enclosed spacer (E).

FIGS. 8 and 9 illustrate certain embodiments of plates andenclosed-spacers (well) for sample thickness regulation. Two exemplaryembodiments are shown: (a) the first plate has an enclosed-spacer (well)and at least one spacer inside the well (FIG. 9 ), and (b) the firstplate does not have a spacer inside the well (FIG. 8 ). Anotherembodiments is that before the first and second plates are in the closedconfiguration, the enclosed spacer is on one of the plate and theisolated spacer(s) are on another plate; and at the closed configurationof the plates, the isolated spacer(s) are inside of the well.

In one embodiment, the volume of the sample deposited on a well of theplate can have a predetermined volume (i.e. meter the volume to aspecific volume) that is about equal to the inner volume of the well(i.e. inner well area times the well height), so that when the platesare in a closed configuration, the sample is nearly completely fill upthe well, with no or nearly no sample flow out of the well.

In another embodiment, the volume of the sample deposited in a well ofthe plate are not metered, and at a closed configuration of the plate, apart of the sample fills up the well nearly completely, while the otherpart of the sample flow out of the well.

In another embodiments, a plurality of the wells are one plate. In someembodiments, there are trenches (dumping spaces) between wells for thesamples that overflow from the wells. The dumping spaces prevent thesample overflow from one well flows into other well(s).

E1. As illustrated in FIG. 8 , a method for assaying and/or chemicalreactions with a small volume sample in a wide well, comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        the first plate has, on its surface, a well that has a        predetermined dimension (including well bottom, depth, and rim)        and a binding site at the bottom of the well;    -   (b) obtaining a sample that (i) contains target entity capable        of binding to the binding site and diffusing in the sample,        and (ii) has a volume and a wetting property such that the        contact area of the sample deposited on only the bottom of the        well, without contacting the second plate, is less than the area        of the well bottom;    -   (c) depositing, when the plates are configured in an open        configuration, the sample inside the well or on a corresponding        area on the second plate, or both; wherein, in the open        configuration: the two plates are partially or completely        separated apart, and the spacing between the second plate and        the bottom of the well is not regulated by the rim of the well        (i.e. the depth of the well);    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration; wherein, in the closed configuration:        the second plate covers over the well, the thickness of the        sample on the binding site is regulated by the well and the        second plate, and the sample has a larger contact area to the        well bottom than that when the plates are in the open        configuration;        -   wherein the corresponding area of the second plate is the            area that is on top of the well and inside the rime of the            well at the closed configuration.

In the method of paragraph E1, the plate further comprises at least anisolated spacer inside the well (i.e. well spacer).

In the method of paragraph E1, in some embodiments, the volume of thesample is metered (e.g. to have a selected volume). The metered volumeis approximately equal to, less than, or larger than the volume of thewell.

In the method of paragraph E1, in some embodiments, a compression forcefrom outside of the plates is configured to hold the plates in theclosed configuration.

In the method of a paragraph E1, in some embodiments, a capillary forceis configured to hold the plates in the closed configuration.

As illustrated in FIG. 8 d , in the method of paragraph E1, in someembodiments, the bottom of the well, the corresponding area of thesecond, or both are attached with spacers of predetermined heights,wherein at the closed configuration the sample thickness is regulated bythe spacers, the rim, or both.

In some embodiments, the spacer height is equal to, less than, or largerthan the depth of the well. The well bottom is planer (i.e. flat) orcurved. In some embodiments, the spacers (1) have a predetermined interspacer spacing, (2) inside a sample, (3) fixed to respective plates, orany combination of thereof.

In some embodiments, the volume of the sample is approximately equal tothe volume of the well minus the volume of the spacers. In someembodiments, the second plate is configured to seal off the well.

In some embodiments, the ratio of the well area to the well depth squareis 3 or larger, 5 or larger, 10 or larger, 20 or larger, 30 or larger,50 or larger, 100 or larger, 200 or larger, 1000 or larger, 10,000 orlarger, or a range between any two of the values.

The ratio of the well area to the well depth square is between 3 and 20in a preferred embodiment, 20 and 100 in another preferred embodiment,and 100 and 1000 in another preferred embodiment, and 1000 and 10,000 inanother preferred embodiment.

17 Quantification by Correcting Effects Generated by None-Sample Volume(C)

In a CROF process, often a sample is mixed with a none-sample-volume(s)which is due to objects that are not the sample, that include, but notlimited to, spacers, air bubbles, dusts, or any combinations of thereof.The air bubbles or dust can be introduced using the sample deposition orother process in the CROF process. These none-sample objects occupyvolume and inside the sample, which should be corrected in determine arelevant volume (a volume of interest) of a sample. One aspect of thepresent invention is to correct the effects generated by the none-samplevolume inside a relevant volume of the sample between two plates, wherethe thickness of the relevant volume is regulated by spacers.

C1. A method for correcting the effects generated by a none-samplematerial in determining a relevant volume of a sample between twoplates, comprising:

-   -   (a) obtaining a sample, wherein a relevant volume of the sample        is to be quantified;    -   (b) obtaining two plates that are movable relative to each other        into different configurations, wherein one or both of the plates        comprise spacers and the spacers have a predetermined        inter-spacer distance and height, and each of the spacers is        fixed with its respective plate;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (d) after (c), bringing the plates into a closed configuration,        wherein, in the closed configuration: the plates are facing each        other, the spacers and the relevant volume of the sample are        between the plates, the thickness of the relevant volume of the        sample is regulated by the plates and the spacers and is thinner        than the maximum thickness of the sample when the plates are in        the open configuration, and the relevant volume may contain a        volume of a none-sample material;    -   (e) measuring, while the plates are in the closed        configuration, (i) the lateral area of the relevant volume of        the sample and (ii) the volume of the none-sample material; and    -   (f) calculating the relevant volume of the sample by using the        thickness of the relevant volume regulated by the spacers and        correcting the effects of a none-sample material;        wherein the relevant volume is at least a portion of an entire        volume of the sample, and the none-sample materials are the        materials that are not from the sample.    -   the measuring of the none-sample volume is by imaging of the        sample between the two plates.

18 Precision Quantification by Double Checking the Spacing

In a CROF, for a given set of conditions, even the spacers and theplates can give a predetermining sample thickness at a closedconfiguration, the actual set of conditions during a particular CROF maybe different from the expected, which lead to errors in thepredetermined final sample thickness. To reduce such errors, one aspectof the present invention is to double check the final sample thicknessat a closed configuration.

C2. A method for determining and checking a thickness of a relevantvolume of a sample between two plates, comprising:

-   -   (a) obtaining a sample, wherein a relevant volume of the sample        is to be quantified;    -   (b) obtaining two plates that are movable relative to each other        into different configurations, wherein one or both of the plates        comprise spacers and the spacers have a predetermined        inter-spacer distance and height, and each of the spacers is        fixed with its respective plate;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (d) after (c), bringing the plates into a closed configuration,        wherein, in the closed configuration: the plates are facing each        other, the spacers and the relevant volume of the sample are        between the plates, the thickness of the relevant volume of the        sample is regulated by the plates and the spacers and is thinner        than the maximum thickness of the sample when the plates are in        the open configuration, and the relevant volume may contain a        volume of a none-sample material;    -   (e) measuring, while the plates are in the closed        configuration, (i) the lateral area of the relevant volume of        the sample and (ii) the volume of the none-sample material; and    -   (f) calculating the relevant volume of the sample by correcting        the effects of a none-sample material;        wherein the relevant volume is at least a portion of an entire        volume of the sample, and the none-sample materials are the        materials that are not from the sample.

19 Wash (WS)

In the present invention, one or any combinations of the embodiments ofthe plate pressing and holding described herein are used in all themethods and devices described in the entire description of the presentinvention.

A method for a wash step in assaying, comprising:

-   -   (a) Performing the steps in one or any combination of the        methods described in above and    -   (b) washing away the sample or the transfer media between the        plates.        In the method that uses CROF, the wash is performed by keep the        plates in the closed-configuration.

In the method that uses CROF, the wash is performed by separating theplates from the closed-configuration.

20 Assays with Multiple Steps (MA)

In the present invention, the embodiments descripted by the disclosures(i.e. all sections) can be used in a combined (a) by combining oneembodiment with other embodiment(s), by using the same embodiment(s)more than one times, and (c) any combination of (a) and (b).

MA1. A method for assaying an analyte in a sample, comprising:

-   -   (a) obtaining a sample with an analyte;    -   (b) performing the method that uses CROF; and    -   (c) separating the plates and performing the method that uses        CROF.

In the method of paragraph MA1, in some embodiments, it furthercomprises, after the step (c) of MA1, a step of repeating the same stepsof all the steps in the method of MA1 at least once.

MA2. A method for assaying an analyte in a sample, comprising:

-   -   (a) obtaining a sample with an analyte;    -   (b) performing the method that uses CROF;    -   (c) separating the plates and performing the method (washing)        that uses CROF; and    -   (d) performing the method that uses CROF.

In the method of paragraph MA2, in some embodiments, it furthercomprises, after the step (d) in MA2, a step of repeating the same stepsof all the steps in the method of MA2 at least once.

In the method of paragraph MA2, in some embodiments, it furthercomprises, after the step (c) in MA2, a step of repeating the same stepsof all the steps in the method of MA1 at least once.

MA3. A kit for assaying an analyte in a sample, comprising:

-   -   a first CROF device that uses CROF; and    -   a third plate that, when the plates of the first CROF device are        separated, combines with one of the plates of the first CROF        device to form a second CROF device.        MA4. A kit for assaying an analyte in a sample, comprising:    -   a first CROF device that uses CROF;    -   at least one binding site or storage site that is on the sample        contact area of the plate of a CROF device; and    -   a third plate that, when the plates of the first CROF device are        separated, combines with one of the plates of the first CROF        device to form a second CROF device;        wherein the binding site binds a target analyte to the plate        surface, and the storage site has a reagent that, upon in touch        with the sample, can be dissolved into the sample and diffuse in        the sample.

The imaging may comprise a use of a smart phone. The methods of thissection may further comprise a step of illumination by a light source.The light source may be a laser, LED, a lamp, or a camera flash light.

A Kit (MQXA) for Performing Assay for Detecting a Target Entity in aSample

A kit for assaying a target entity in a sample, may comprise:

a. a first plate, wherein one surface of the first plate has one or aplurality of binding site(s) that can immobilize a target entity and thebinding site has binding partner that binds the target entity;

b. a cover plate;

c. a sample in the inner space between the cover plate and the firstplate, wherein the sample contains said target entity that is mobile inthe sample, the shape of sample is deformable, the first plate and thesecond plate are movable relative to each other, the shape of the sampleis substantially conformal to the inner surfaces, at least a part of thesample is in contact to the binding site, and the inner spacing is,during incubation, less than certain distance. the sample is in contactwith said binding sites;

d. an imaging device that can image the first plate surface and/or thecover plate surface; and

e. a measuring device that can measure the spacing of the inner space.

The methods of this section may include use of a smart phone. Themethods of this section may include use of an illuminating device. Theilluminating device may comprise a laser, LED, a lamp, or a camera flashlight.

21 Plate Pressing and Holding (H)

Compressing forces. In a CROF process, forces are used to compress thetwo plates to bring the plates from an open configuration to a closedconfiguration. The compressing forces reduce the spacing between theinner surfaces of the plates and hence a thickness of the sample that isbetween the plates. In the present invention, the compressing forcesinclude, but not limited to, mechanical force, capillary forces (due tosurface tensions), electrostatic force, electromagnetic force (includingthe light), and any combination of thereof.

In some embodiments of bring the plates from an open configuration to aclosed configuration, an external force is applied to push the firstplate and the second plate to toward each other.

In some embodiments of bring the plates from an open configuration to aclosed configuration, an external pressure is applied to outside thefirst plate and the second plate to push the plates toward each other,and the pressure is higher than the pressure inside of the plate. Adevice is used to make the pressure of outside the plates higher thanthat inside the plate. The device include, in limited to, a sealingdevice.

In some embodiments, the compress force is at least partially providedby the capillary force, which is due to a liquid between the first plateand the second plate and the corresponding surface tensions andinteractions with the plates. In some embodiments, the liquid is thesample itself, or the sample mixed with liquid. In certain embodiments,capillary force is used together with other forces. In many cases, asample is often in liquid and the surface tensions are suited forinserting a capillary force. In some embodiments, the sample deformationby the plates can automatically stop when the capillary force equals tothe force needed to deform the sample.

In certain embodiments, the compressing force (hence the sampledeformation) is created by isolating the pressure between the firstplate and the second plate (inside pressure) from that outside of theplates (outside pressure), and then make the inside pressure lower thanthe outside pressure. The isolation can be done using a vacuum seal orother devices.

In some embodiments, it is a combination of the methods described above.

Gradual Pressing. In certain embodiments, the compressing force to bringthe plates to a closed configuration is applied in a process, termed“gradual pressing”, which comprises: pressing (i.e. applying thecompressing the force) is applied at one location of the plate(s) first,then is applied gradually to other locations of the sample. In someembodiments of the gradual pressing, the compressing force (except thecapillary forces by the sample itself) at one location is, afterdeformed the sample to a desired thickness at that location, (i)maintained during the entire process of the pressing and the sampledeformation, (ii) removed while other locations being pressed, or (iii)a use of (i) for certain part of the plates and a use of (ii) for otherpart of the sample.

In one embodiment of the gradual pressing, a roller is being used topress the first plate and the second plate (the sample is between theplates, and the plates are slightly flexible) against another roller ora flat surface.

In another embodiment, the human fingers are the tool of the pressingthe plates (hence the sample). The pressing is one part of human handagainst another part of human body (including another part of humanhand) or a human hand against an object (e.g. a table surface). In oneembodiment, the pressing starts at one location of the sample andgradual moved to other locations of the sample.

In one embodiment of the gradual pressing, a pressed air jet is firstdirected to a location (e.g. the center) of the plate pair (which isbetween the first plate and the second plate, one of the plates isslightly flexible) and the pressure is gradually extended to other partof the plate pair.

In another embodiment, one or both of the first plate and the secondplate is flexible and is in contact with one location of the sample,then a capillary force in that location pulls the plate pair together(toward to each other) to deform the sample.

Advantage of the gradual pressing include: it allows one to use lessforce to deform the sample (because for the same force, the smallerpress area, the larger the pressure); it helps motion (deformation) ofthe sample, and/or it reduces air bubble in the sample. The largerpressure is, the more sample deformation will be. A gradual pressing canimprove the thickness uniformity of the deformed sample.

Pressing devices. The devices for asserting the compressing force(s) forthe sample deformation in CROF have several implementations. Someembodiments are to use human hand to press, for example, to press byhuman fingers. Certain embodiments are to use a press device, where thepress device includes, but not limited to, a human hand(s), a mechanicalclip, a mechanical press, mechanical clamp, a mechanical slider, amechanical device, ab electromagnetic device, roller that rolls on asurface, two rollers against each other, fluidic press, a hydraulicdevice, or any combination of thereof. Certain embodiments are usepressured liquid (including pressed air) to press the first plate and/orthe second plate directly or indirectly. “Directly” means the pressuredliquid is applied directly on the first plate and/or the second plate;and the “indirectly” means it is applied through a third object. Certainembodiments in pressing use a combination of the above embodiments ofpressing devices and methods.

Furthermore, in some embodiments of the sample deformation, the pressingand the sample deformation are monitored. The monitoring can be used tocontrol the pressing and the sample deformation. The monitoring of thedeformation include, but not limited to, a mechanical method,electrical, optical, chemical, magnetic, and any combination of thereof.The mechanical methods include, but not limited to, mechanical gauges,spacer (mechanical stoppers, more discussed below), and sound waves.

In CROF, the spacing control device comprises mechanical press,mechanical translation stages, human fingers, liquid that providecapillary forces that pulls the plates toward each other, liquid(including air) that applies a pressure on the plates, or a combinationof thereof.

In certain embodiments, the mechanical stages (translational and/orrotational) are used for the sample deformation and sample thicknesscontrol and work together with the monitoring systems.

In some embodiments, the compressing force is at least partly suppliedby a press (which is a device that bring the plates to a closedconfiguration) configured to press the plates together into the closedconfiguration.

In some embodiments, the plate pressing is to use a human hand. Thehuman can be the person being tested or a person who perform the test,or a person who collecting the sample.

In some embodiments, the plate pressing is to hold the two platestogether is to use a capillary force. The capillary force is generatedby making at least a portion of the inner surface of one plate or bothhydrophilic. With a proper capillary force, the two plates is able tomaintain the same plate-spacing and the same thickness of the relevantvolume of the sample as that when the plates initially in the closedconfiguration, even a part or all of the forces (except the capillaryforce) that were used to compress the plate to the close configurationis removed.

In some embodiments, the device that applies a compressing force on theouter surface of the plates to reducing the plate inner surface spacingcomprise a contacting surface that is comfortable to the outer surfacesof the plate, wherein the contacting surface of the device is thesurface of the device that contacts the outer surface of the plates, andthe “conformable to the outer surface of the plate” means that thedevice surface can deform, during the compressing, it shape to conformthe shape of the plate outer surface. In one exemplary embodiment, thecompressing device is human figures. In another exemplary embodiment,the compressing device has a contacting surface made of soft plastics orrubbers.

Self-holding (maintaining the final sample thickness after removingcompressing forces). In some embodiments of pressing in CROF, after thesample deformation at a closed configuration, some of the compressingforces are removed and the sample maintains the same final samplethickness as the compression forces still exist. Such situation istermed “self-holding”. One reason for self-holding is that afterremoving the compressing forces that were inserted from outside of theplate pair, there are still other forces exist between the innersurfaces of the plates, such as a capillary force, which hold the platepair together. The capillary force is the due to the wetting propertiesof the sample on the plates.

To have self-holding, one needs to control the plate surface wettingproperties, the total contact area of the sample to the plates, thefinal sample thickness at a closed configuration, or a combination ofthereof.

In some embodiments to achieve self-holding, one or both inner surfacesof the plates is hydrophilic. Namely, it is either one of plates have aninner surface that is hydrophilic or both of the plates have an innersurface that is hydrophilic.

The capillary force depends on the radius curvature of the liquidsurface, smaller the curvature and higher the capillary force. A smallercurvature can be achieved by using smaller spacing between the twoplates (i.e. plate pair) and hence a smaller sample thickness. In someembodiments, a final sample thickness for achieving self-holding is 10nm or less, 100 nm or less, 100 nm or less, 500 nm or less, 1 um(micrometer) or less, 2 um or less, 3 um or less, 5 um or less, 10 um orless, 20 um or less, 50 um or less, 70 um or less, 100 um or less, 150um or less, 300 um or less, 500 um or less, 700 um or less, 1000 um orless, 1200 um or less, or a range between any two of the values.

In some embodiments, the area of the sample in contract with the platesfor self-holding is at most 10 um², at most 100 um², at most 200 um², atmost 500 um², at most 1000 um², at most 2000 um², at most 5000 um², atmost 8,000 um², at most 0.01 mm², at most 0.05 mm², at most 0.1 mm², atmost 0.5 mm², at most 1 mm², at most 5 mm², at most 10 mm², at most 50mm², at most 100 mm², at most 500 mm², at most 1,000 mm², at most 2,000mm², at most 5,000 mm², at most 10,000 mm², at most 100,000 mm², or arange between any two of the values.

In some embodiments, one or both of the plate inner surface's wettingproperties is modified for better self-holding.

HS.1 In some embodiments, in a CROF process, a device is used to inserta compressing force to bring the plates into a closed configuration, andafter the closed configuration is reached, the compressing force by thedevice is removed and the sample thickness and the inner surface spacingof the plates are remained approximately the same as that beforeremoving the compressing force by the device. In some embodiments, inthe methods of previous paragraph, it further comprises a step ofreading a signal from the plates or between the plates, wherein thesignal includes, but not limited to, a signal related to analytes,entity, labels, sample volume, concentration of a matter (i.e.chemicals), or any combination of thereof.

In the method of paragraph SH.1, the device is a human hand(s), amechanical clip, a mechanical press, mechanical clamp, a mechanicalslider, a mechanical device, ab electromagnetic device, roller thatrolls on a surface, two rollers against each other, fluidic press, ahydraulic device, or any combination of thereof.

In the method of paragraph SH.1, in some embodiments, “the samplethickness and the inner surface spacing of the plates are remainedapproximately the same as that before removing the compressing force bythe device” means that the relative difference of the sample thicknessand the plate inner surface spacing before and after removing thecompressing force is 0.001% or less, 0.01% or less, 0.1% or less; 0.5%or less, 1% or less, 2% or less, 5% or less, 8% or less, 10% or less,15% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% orless, 70% or less, 80% or less, 90% or less, 99.9% or less, or a rangebetween any of the values.

In the method of paragraph SH.1, in some embodiments, the samplethickness and the inner surface spacing of the plates after removing thecompressing force by the device care predetermined, whereinpredetermined means that the thickness and the spacing after removingthe compressing force is known before applying the compressing force fora given compressing conditions.

H1. A method for reducing the thickness of a relevant volume of a sampleand maintain the reduced thickness, comprising:

-   -   (a) obtaining a sample, wherein a thickness of a relevant volume        of the sample is to be reduced;    -   (b) obtaining two plates that are movable relative to each other        into different configurations, wherein one or both of the plates        comprise spacers and the spacers have a predetermined        inter-spacer distance and height, and each of the spacers is        fixed with its respective plate;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are either partially or completely separated apart and        the spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by using a pressing device        that brings the plates into a closed configuration, wherein, in        the closed configuration: the plates are facing each other, the        spacers and the relevant volume of the sample are between the        plates, the thickness of the relevant volume of the sample is        regulated by the plates and the spacers and is thinner than the        maximum thickness of the sample when the plates are in the open        configuration, and at least one of the spacers is inside the        sample; and    -   (e) after (d), releasing the device, wherein after releasing the        pressing device, the spacing between the plates remains the same        as or approximately same as that when the device is applied.        -   wherein the relevant volume is at least a portion of an            entire volume of the sample.

In the method of paragraph H1, the approximately same as the spacingbetween the plates is at most 1%, at most 2%, at most 5%, at most 10%,at most 20%, at most 50%, at most 60%, at most 70%, at most 80%, at most90%, or a range between any two of the values.

For example, in CROF, a human hand or hands are used to compressed twoplate to a closed position, then the hand(s) and hence the compressingforce by hand(s) are removed, but the final sample thickness is stillthe same as that when the compressing force by hands exist.

22 Other Combinations

In the present invention, each of the embodiments in the disclosures(i.e. all sections) can be used (a) alone, (b) combined with otherembodiment(s), (c) multiple times, and (d) any combination of (a) to(c).

The methods and devices in the present invention disclosed can be usedalone or any combination of thereof. The term a “AMAX” method or devicerefers to a method or device of the embodiments described here.

In some embodiments, the methods and devices in the present inventiondisclosed can be used in the form of Q, X, A, M, QX, QA, QM, XA, XM, AM,QXA, QAM, XAM, and QXAM.

Some embodiments of application of the Q, X, A, and M to surfaceimmobilization assay, comprising

a. having a first plate, wherein the first plate surface has at leastone well of a known depth and volume, and bottom surface of the well hasone or a plurality of binding site(s) that can immobilize a targetentity in a sample;

b. depositing, into the well, the sample of a volume approximately thesame as the well volume, wherein the sample contains the targetedentity, the targeted entity is mobile in the sample, the shape of sampleis deformable, and the sample covers only a part of the well (hence havea simple thickness higher than the well depth);

c. having a cover plate;

d. facing the first plate and the cover plate to each other, wherein thesample is between the inner surfaces of the first plate and the secondplate;

e. reducing the sample thickness by reducing the spacing between theinner surfaces of the first plate and the second plate; and

f. Incubating the sample at the reduced sample thickness for a period oftime;

One variation of these methods is to apply one or more of the abovesteps to 96 well plates or other well plates.

The methods and devices in the present invention disclosed in Section 1,2, 3, and 5, can be used alone or any combination of thereof.Specifically, we use Q for the inventions disclosed in Section 1 and 2,A for the inventions disclosed in Section 3 and 5, X for the inventionsdisclosed in Section 4 and 5, and M for the inventions disclosed inSection 6. Hence the methods and devices in the present inventiondisclosed in Section 1, 2, 3, and 5, can be used in the form of Q, X, A,M, QX, QA, QM, XA, XM, AM, QXA, QAM, XAM, and QXAM.

Some embodiments of application of the Q, X, A, and M to surfaceimmobilization assay, comprising

a. having a first plate, wherein the first plate surface has at leastone well of a known depth and volume, and bottom surface of the well hasone or a plurality of binding site(s) that can immobilize a targetentity in a sample;

b. depositing, into the well, the sample of a volume approximately thesame as the well volume, wherein the sample contains the targetedentity, the targeted entity is mobile in the sample, the shape of sampleis deformable, and the sample covers only a part of the well (hence havea simple thickness higher than the well depth);

c. having a cover plate;

d. facing the first plate and the cover plate to each other, wherein thesample is between the inner surfaces of the first plate and the secondplate;

e. reducing the sample thickness by reducing the spacing between theinner surfaces of the first plate and the second plate; and

f. Incubating the sample at the reduced sample thickness for a period oftime.

One variation of these methods is to apply one or more of the abovesteps to 96 well plates or other well plates.

Several embodiments of the methods, devices, and systems combine one ormore of the features of sample volume quantification (Q), reagentsaddition (A), and/or assay acceleration (X) (and may be referred to asthe corresponding acronyms QA, QX, AX, and QAX). Some experimentaldemonstrations of Q, A, X, QA, QX, AX, and QAX methods and devices aredescribed below.

23 Reagents

The term “reagents” refers to, unless stated otherwise, one or more ofbiological agents, biochemical agents, and/or chemical agents. Forexample, reagents may include capture agents, detection agents, chemicalcompounds, optical labels, radioactive labels, enzymes, antibodies,proteins, nucleic acids, DNA, RNA, lipids, carbohydrates, salts, metals,surfactants, solvents, or any combination of thereof.

In some embodiments, the reagents on a plate in the form of liquid,solid, molecular vapor, or a combination of thereof. The deposition ofreagent, include, but are not limited to, depositing, placing, printing,stamping, liquid dispensing, evaporation (thermal evaporation, vaporevaporation, human breathing), chemical vapor deposition, and/orsputtering. Different reagents can be in different locations. Reagentsmay be printed and/or deposited as small dots of reagents.

In some embodiments, the reagents are deposited on a plate in a liquidor vapor form first, then are dried to become dry reagents on the platebefore a CROF process.

Controlling Reagents Releasing Time. A-methods may further comprise astep of controlling the reagent release time (i.e. the time measures howfast a reagent can be dissolved in a sample. Some embodiments incontrolling the reagent release time of a reagent comprises a step ofmixing or coating on top of the reagent a or several “releasing controlmaterial(s)” that affect the release (into the sample) of the reagent.In some embodiments, the releasing control material can be anotherreagent. For example, there are two reagents A and B, the reagent A iscoated on top of the reagent B, under certain conditions, the reagent Awill be dissolved into the sample before the reagent B.

Furthermore, the surface properties of the first plate and the secondplate may be used to control the reagent release. One example is tocontrol the surface wetting properties. For many reagents, a hydrophobicsurface binds the reagent well, hence leading to slow release or norelease of the reagent into the sample (depending upon how thick is thereagent layer), while a hydrophilic surface binds the reagent poorlyhence leading a fast release into the sample.

Drying of Reagents. In some embodiments, after the reagent depositionstep (c) but before the sample deposition step (d), A-methods furthercomprise a step of drying some or all of the reagents deposited in thestep (c).Location of Reagents. Reagents may be applied and/or arranged on one orboth of the plates. Reagents may be in storage sites (locations) on theplate(s), with each storage site including one or more reagents.Different storage sites may include different reagents, the samereagents, or one or more common reagents.

Control Concentration of Added Reagents. In some embodiments, themethods may further comprise a step of controlling the concentration ofthe added reagents by controlling the samples thickness over the storagesites (i.e., the surface with reagents).

The reagent used in the present invention may be any suitable reagentrequired for an assay, e.g., a labeled or unlabeled antibody, a labeledor unlabeled nucleic acid, an enzyme that may or may not contain anaffinity moiety, etc. In some embodiments and as noted above, the storedreagent may be a component of an assay designed to test a blood or otherliquid sample for the presence of an analyte. For example, chloride ionscan be measured by any of the following protocols, and components ofthese assays may be present in a storage site: Colorimetric methods:chloride ions displace thiocyanate from mercuric thiocyanate. Freethiocyanate reacts with ferric ions to form a colored complex-ferricthiocyanate, which is measured photometrically. Coulometric methods:passage of a constant direct current between silver electrodes producessilver ions, which react with chloride, forming silver chloride. Afterall the chloride combines with silver ions, free silver ions accumulate,causing an increase in current across the electrodes and indicating theend point to the reaction. Mercurimetric methods: chloride is titratedwith a standard solution of mercuric ions and forms HgCl2 solublecomplex. The end point for the reaction is detected colorimetricallywhen excess mercury ions combine with an indicator dye,diphenylcarbazone, to form a blue color. Likewise, magnesium can bemeasured colorimetrically using calmagite, which turns a red-violetcolor upon reaction with magnesium; by a formazan dye test; emits at 600nm upon reaction with magnesium or using methylthymol blue, which bindswith magnesium to form a blue colored complex. Likewise, calcium can bedetected by a colorimetric technique using O-Cresolphtalein, which turnsa violet color upon reaction of O-Cresolphtalein complexone withcalcium. Likewise, Bicarbonate cab ne tested bichromatically becausebicarbonate (HCO3⁻) and phosphoenolpyruvate (PEP) are converted tooxaloacetate and phosphate in the reaction catalyzed byphosphoenolpyruvate carboxylase (PEPC). Malate dehydrogenase (MD)catalyzes the reduction of oxaloacetate to malate with the concomitantoxidation of reduced nicotinamide adenine dinucleotide (NADH). Thisoxidation of NADH results in a decrease in absorbance of the reactionmixture measured bichromatically at 380/410 nm proportional to theBicarbonate content of the sample. Blood urea nitrogen can be detectedin a colorimetric test in which diacetyl, or fearon develops a yellowchromogen with urea and can be quantified by photometry, or multiusingthe enzyme urease, which converts urea to ammonia and carbonic acid,which can be assayed by, e.g., i) decrease in absorbance at 340 nm whenthe ammonia reacts with alpha-ketoglutaric acid, ii) measuring the rateof increase in conductivity of the solution in which urea is hydrolyzed.Likewise, creatinine can be measured colorimetrically, by treated thesample with alkaline picrate solution to yield a red complex. Inaddition, creatine can be measured using a non-Jaffe reaction thatmeasures ammonia generated when creatinine is hydrolyzed by creatinineiminohydrolase. Glucose can be measured in an assay in which blood isexposed to a fixed quantity of glucose oxidase for a finite period oftime to estimate concentration. After the specified time, excess bloodis removed and the color is allowed to develop, which is used toestimate glucose concentration. For example, glucose oxidase reactionwith glucose forms nascent oxygen, which converts potassium iodide (inthe filter paper) to iodine, forming a brown color. The concentration ofglycosylated hemoglobin as an indirect read of the level of glucose inthe blood. When hemolysates of red cells are chromatographed, three ormore small peaks named hemoglobin A1a, A1b, and A1c are eluted beforethe main hemoglobin A peak. These “fast” hemoglobins are formed by theirreversible attachment of glucose to the hemoglobin in a two-stepreaction. Hexokinase can be measured in an assay in which glucose isphosphorylated by hexokinase (HK) in the presence of adenosinetriphosphate (ATP) and magnesium ions to produce glucose-6-phosphate andadenosine diphosphate (ADP). Glucose-6-phosphate dehydrogenase (G6P-DH)specifically oxidises glucose-6-phosphate to gluconate-6-phosphate withthe concurrent reduction of NAD+ to NADH. The increase in absorbance at340 nm is proportional to the glucose concentration in the sample. HDL,LDL, triglycerides can be measured using the Abell-Kendall protocol thatinvolves color development with Liebermann-Burchard reagent (mixedreagent of acetic anhydride, glacial acetic acid, and concentratedsulfuric acid) at 620 nm after hydrolysis and extraction of cholesterol.A fluorometric analysis may be used utilized to determine triglyceridereference values. Plasma high-density lipoprotein cholesterol (HDL-C)determination is measured by the same procedures used for plasma totalcholesterol, after precipitation of apoprotein B-containing lipoproteinsin whole plasma (LDL and VLDL) by heparin-manganese chloride. Thesecompounds can also be detected colorimetrically in an assay that isbased on the enzyme driven reaction that quantifies both cholesterolesters and free cholesterol. Cholesterol esters are hydrolyzed viacholesterol esterase into cholesterol, which is then oxidized bycholesterol oxidase into the ketone cholest-4-en-3-one plus hydrogenperoxide. The hydrogen peroxide is then detected with a highly specificcolorimetric probe. Horseradish peroxidase catalyzes the reactionbetween the probe and hydrogen peroxide, which bind in a 1:1 ratio.Samples may be compared to a known concentration of cholesterolstandard.

24 Applications, Samples, and More Bio/Chemical Biomarkers

The applications of the present invention include, but not limited to,(a) the detection, purification and quantification of chemical compoundsor biomolecules that correlates with the stage of certain diseases,e.g., infectious and parasitic disease, injuries, cardiovasculardisease, cancer, mental disorders, neuropsychiatric disorders andorganic diseases, e.g., pulmonary diseases, renal diseases, (b) thedetection, purification and quantification of microorganism, e.g.,virus, fungus and bacteria from environment, e.g., water, soil, orbiological samples, e.g., tissues, bodily fluids, (c) the detection,quantification of chemical compounds or biological samples that posehazard to food safety or national security, e.g. toxic waste, anthrax,(d) quantification of vital parameters in medical or physiologicalmonitor, e.g., glucose, blood oxygen level, total blood count, (e) thedetection and quantification of specific DNA or RNA from biosamples,e.g., cells, viruses, bodily fluids, (f) the sequencing and comparing ofgenetic sequences in DNA in the chromosomes and mitochondria for genomeanalysis or (g) to detect reaction products, e.g., during synthesis orpurification of pharmaceuticals. The present invention also can be usedin the various fields include, but not limited to, human, veterinary,agriculture, foods, environments, drug testing, and others.

The detection can be carried out in various sample matrix, such ascells, tissues, bodily fluids, and stool. Bodily fluids of interestinclude but are not limited to, amniotic fluid, aqueous humour, vitreoushumour, blood (e.g., whole blood, fractionated blood, plasma, serum,etc.), breast milk, cerebrospinal fluid (CSF), cerumen (earwax), chyle,chime, endolymph, perilymph, feces, gastric acid, gastric juice, lymph,mucus (including nasal drainage and phlegm), pericardial fluid,peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil),semen, sputum, sweat, synovial fluid, tears, vomit, urine and exhaledcondensate. In some embodiments, the sample comprises a human bodyfluid. In some embodiments, the sample comprises at least one of cells,tissues, bodily fluids, stool, amniotic fluid, aqueous humour, vitreoushumour, blood, whole blood, fractionated blood, plasma, serum, breastmilk, cerebrospinal fluid, cerumen, chyle, chime, endolymph, perilymph,feces, gastric acid, gastric juice, lymph, mucus, nasal drainage,phlegm, pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum,saliva, sebum, semen, sputum, sweat, synovial fluid, tears, vomit,urine, and exhaled condensate.

In embodiments, the sample is at least one of a biological sample, anenvironmental sample, and a biochemical sample.

In some embodiments, the sample is at least one of a biological sample,an environmental sample, and a biochemical sample.

In any embodiment, the CROF device may be placed in a microfluidicdevice and the applying step b) may include applying a sample to amicrofluidic device comprising the CROF device.

In any embodiment, the reading step d) may include detecting afluorescence or luminescence signal from the CROF device.

In any embodiment, the reading step d) may include reading the CROFdevice with a handheld device configured to read the CROF device. Thehandheld device may be a mobile phone, e.g., a smart phone.

In any embodiment, the CROF device may include a labeling agent that canbind to an analyte-capture agent complex on the CROF device.

In any embodiment, the devices, systems and methods in the presentinvention may further include, between steps c) and d), the steps ofapplying to the CROF device a labeling agent that binds to ananalyte-capture agent complex on the CROF device, and washing the CROFdevice.

In any embodiment, the reading step d) may include reading an identifierfor the CROF device. The identifier may be an optical barcode, a radiofrequency ID tag, or combinations thereof.

In any embodiment, the devices, systems and methods in the presentinvention may further include applying a control sample to a controlCROF device containing a capture agent that binds to the analyte,wherein the control sample includes a known detectable amount of theanalyte, and reading the control CROF device, thereby obtaining acontrol measurement for the known detectable amount of the analyte in asample.

In any embodiment, the sample may be a diagnostic sample obtained from asubject, the analyte may be a biomarker, and the measured amount of theanalyte in the sample may be diagnostic of a disease or a condition.

The amount of sample may be about a drop of a sample. The amount ofsample may be the amount collected from a pricked finger or fingerstick.The amount of sample may be the amount collected from a microneedle or avenous draw.

A sample may be used without further processing after obtaining it fromthe source, or may be processed, e.g., to enrich for an analyte ofinterest, remove large particulate matter, dissolve or resuspend a solidsample, etc.

Any suitable method of applying a sample to the CROF device may beemployed. Suitable methods may include using a pipet, dropper, syringe,etc. In certain embodiments, when the CROF device is located on asupport in a dipstick format, as described below, the sample may beapplied to the CROF device by dipping a sample-receiving area of thedipstick into the sample.

A sample may be collected at one time, or at a plurality of times.Samples collected over time may be aggregated and/or processed (byapplying to a CROF device and obtaining a measurement of the amount ofanalyte in the sample, as described herein) individually. In someinstances, measurements obtained over time may be aggregated and may beuseful for longitudinal analysis over time to facilitate screening,diagnosis, treatment, and/or disease prevention.

Washing the CROF device to remove unbound sample components may be donein any convenient manner, as described above. In certain embodiments,the surface of the CROF device is washed using binding buffer to removeunbound sample components.

Detectable labeling of the analyte may be done by any convenient method.The analyte may be labeled directly or indirectly. In direct labeling,the analyte in the sample is labeled before the sample is applied to theCROF device. In indirect labeling, an unlabeled analyte in a sample islabeled after the sample is applied to the CROF device to capture theunlabeled analyte, as described below.

Data Processing.

In certain embodiments, the subject device is configured to process dataderived from reading the CROF device. The device may be configured inany suitable way to process the data for use in the subject methods. Incertain embodiments, the device has a memory location to store the dataand/or store instructions for processing the data and/or store adatabase. The data may be stored in memory in any suitable format.

In certain embodiments, the device has a processor to process the data.In certain embodiments, the instructions for processing the data may bestored in the processor, or may be stored in a separate memory location.In some embodiments, the device may contain a software to implement theprocessing.

In certain embodiments, a device configured to process data acquiredfrom the CROF device contains software implemented methods to performthe processing. Software implemented methods may include one or more of:image acquisition algorithms; image processing algorithms; userinterface methods that facilitate interaction between user andcomputational device and serves as means for data collection,transmission and analysis, communication protocols; and data processingalgorithms. In certain embodiments, image processing algorithms includeone or more of: a particle count, a LUT (look up table) filter, aparticle filter, a pattern recognition, a morphological determination, ahistogram, a line profile, a topographical representation, a binaryconversion, or a color matching profile.

In certain embodiments, the device is configured to display informationon a video display or touchscreen display when a display page isinterpreted by software residing in memory of the device. The displaypages described herein may be created using any suitable softwarelanguage such as, for example, the hypertext markup language (“HTML”),the dynamic hypertext markup language (“DHTML”), the extensiblehypertext markup language (“XHTML”), the extensible markup language(“XML”), or another software language that may be used to create acomputer file displayable on a video or other display in a mannerperceivable by a user. Any computer readable media with logic, code,data, instructions, may be used to implement any software or steps ormethodology. Where a network comprises the Internet, a display page maycomprise a webpage of a suitable type.

A display page according to the invention may include embedded functionscomprising software programs stored on a memory device, such as, forexample, VBScript routines, JScript routines, JavaScript routines, Javaapplets, ActiveX components, ASP.NET, AJAX, Flash applets, Silverlightapplets, or AIR routines.

A display page may comprise well known features of graphical userinterface technology, such as, for example, frames, windows, scrollbars, buttons, icons, and hyperlinks, and well known features such as a“point and click” interface or a touchscreen interface. Pointing to andclicking on a graphical user interface button, icon, menu option, orhyperlink also is known as “selecting” the button, option, or hyperlink.A display page according to the invention also may incorporatemultimedia features, multi-touch, pixel sense, IR LED based surfaces,vision-based interactions with or without cameras.

A user interface may be displayed on a video display and/or displaypage. The user interface may display a report generated based onanalyzed data relating to the sample, as described further below.

The processor may be configured to process the data in any suitable wayfor use in the subject methods. The data is processed, for example, intobinned data, transformed data (e.g., time domain data transformed byFourier Transform to frequency domain), or may be combined with otherdata. The processing may put the data into a desired form, and mayinvolve modifying the format of data. Processing may include detectionof a signal from a sample, correcting raw data based on mathematicalmanipulation or correction and/or calibrations specific for the deviceor reagents used to examine the sample; calculation of a value, e.g., aconcentration value, comparison (e.g., with a baseline, threshold,standard curve, historical data, or data from other sensors), adetermination of whether or not a test is accurate, highlighting valuesor results that are outliers or may be a cause for concern (e.g., aboveor below a normal or acceptable range, or indicative of an abnormalcondition), or combinations of results which, together, may indicate thepresence of an abnormal condition, curve-fitting, use of data as thebasis of mathematical or other analytical reasoning (includingdeductive, inductive, Bayesian, or other reasoning), and other suitableforms of processing. In certain embodiments, processing may involvecomparing the processed data with a database stored in the device toretrieve instructions for a course of action to be performed by thesubject.

In certain embodiments, the device may be configured to process theinput data by comparing the input data with a database stored in amemory to retrieve instructions for a course of action to be performedby the subject. In some embodiments, the database may contain storedinformation that includes a threshold value for the analyte of interest.The threshold value may be useful for determining the presence orconcentration of the one or more analytes. The threshold value may beuseful for detecting situations where an alert may be useful. The datastorage unit may include records or other information that may be usefulfor generating a report relating to the sample.

In certain embodiments, the device may be configured to receive datathat is derived from the CROF device. Thus in certain cases, the devicemay be configured to receive data that is not related to the sampleprovided by the subject but may still be relevant to the diagnosis. Suchdata include, but are not limited to the age, sex, height, weight,individual and/or family medical history, etc. In certain embodiments,the device is configured to process data derived from or independentlyfrom a sample applied to the CROF device.

Network. In certain embodiments the device may be configured tocommunicate over a network such as a local area network (LAN), wide areanetwork (WAN) such as the Internet, personal area network, atelecommunications network such as a telephone network, cell phonenetwork, mobile network, a wireless network, a data-providing network,or any other type of network. In certain embodiments the device may beconfigured to utilize wireless technology, such as Bluetooth or RTMtechnology. In some embodiments, the device may be configured to utilizevarious communication methods, such as a dial-up wired connection with amodem, a direct link such as TI, integrated services digital network(ISDN), or cable line. In some embodiments, a wireless connection may beusing exemplary wireless networks such as cellular, satellite, or pagernetworks, general packet radio service (GPRS), or a local data transportsystem such as Ethernet or token ring over a LAN. In some embodiments,the device may communicate wirelessly using infrared communicationcomponents.

In certain embodiments, the device is configured to receive a computerfile, which can be stored in memory, transmitted from a server over anetwork. The device may receive tangible computer readable media, whichmay contain instructions, logic, data, or code that may be stored inpersistent or temporary memory of the device, or may affect or initiateaction by the device. One or more devices may communicate computer filesor links that may provide access to other computer files.

In some embodiments, the device is a personal computer, server, laptopcomputer, mobile device, tablet, mobile phone, cell phone, satellitephone, smartphone (e.g., iPhone, Android, Blackberry, Palm, Symbian,Windows), personal digital assistant, Bluetooth device, pager, land-linephone, or other network device. Such devices may becommunication-enabled devices. The term “mobile phone” as used hereinrefers to a telephone handset that can operate on a cellular network, aVoice-Over IP (VoIP) network such as Session Initiated Protocol (SIP),or a Wireless Local Area Network (WLAN) using an 802.11x protocol, orany combination thereof. In certain embodiments, the device can behand-held and compact so that it can fit into a consumer's wallet and/orpocket (e.g., pocket-sized).

Environmental testing. As summarized above, the devices, systems andmethods in the present invention may find use in analyzing anenvironmental sample, e.g., a sample from water, soil, industrial waste,etc., for the presence of environmental markers. An environmental markermay be any suitable marker, that can be captured by a capturing agentthat specifically binds the environmental marker in a CROF deviceconfigured with the capturing agent. The environmental sample may beobtained from any suitable source, such as a river, ocean, lake, rain,snow, sewage, sewage processing runoff, agricultural runoff, industrialrunoff, tap water or drinking water, etc. In some embodiments, thedevices and systems in the present invention detect the concentration oflead or toxins in water. In some embodiments, the presence or absence,or the quantitative level of the environmental marker in the sample maybe indicative of the state of the environment from which the sample wasobtained. In some cases, the environmental marker may be a substancethat is toxic or harmful to an organism, e.g., human, companion animal,plant, etc., that is exposed to the environment. In some cases, theenvironmental marker may be an allergen that may cause allergicreactions in some individuals who are exposed to the environment. Insome instances, the presence or absence, or the quantitative level ofthe environmental marker in the sample may be correlated with a generalhealth of the environment. In such cases, the general health of theenvironment may be measured over a period of time, such as week, months,years, or decades.

In some embodiments, the devices, systems and methods in the presentinvention further includes receiving or providing a report thatindicates the safety or harmfulness for a subject to be exposed to theenvironment from which the sample was obtained based on informationincluding the measured amount of the environmental marker. Theinformation used to assess the safety risk or health of the environmentmay include data other than the type and measured amount of theenvironmental marker. These other data may include the location,altitude, temperature, time of day/month/year, pressure, humidity, winddirection and speed, weather, etc. The data may represent an averagevalue or trend over a certain period (minutes, hours, days, weeks,months, years, etc.), or an instantaneous value over a shorter period(milliseconds, seconds, minutes, etc.).

The report may be generated by the device configured to read the CROFdevice, or may be generated at a remote location upon sending the dataincluding the measured amount of the environmental marker. In somecases, an expert may be at the remote location or have access to thedata sent to the remote location, and may analyze or review the data togenerate the report. The expert may be a scientist or administrator at agovernmental agency, such as the US Centers for Disease Control (CDC) orthe US Environmental Protection Agency (EPA), a research institution,such as a university, or a private company. In certain embodiments, theexpert may send to the user instructions or recommendations based on thedata transmitted by the device and/or analyzed at the remote location.

Foodstuff testing. As summarized above, the devices, systems and methodsin the present invention may find use in analyzing a foodstuff sample,e.g., a sample from raw food, processed food, cooked food, drinkingwater, etc., for the presence of foodstuff markers. A foodstuff markermay be any suitable marker, such as those shown in Table B9, below, thatcan be captured by a capturing agent that specifically binds thefoodstuff marker in a CROF device configured with the capturing agent.The environmental sample may be obtained from any suitable source, suchas tap water, drinking water, prepared food, processed food or raw food,etc. In some embodiments, the presence or absence, or the quantitativelevel of the foodstuff marker in the sample may be indicative of thesafety or harmfulness to a subject if the food stuff is consumed. Insome embodiments, the foodstuff marker is a substance derived from apathogenic or microbial organism that is indicative of the presence ofthe organism in the foodstuff from which the sample was obtained. Insome embodiments, the foodstuff marker is a toxic or harmful substanceif consumed by a subject. In some embodiments, the foodstuff marker is abioactive compound that may unintentionally or unexpectedly alter thephysiology if consumed by the subject. In some embodiments, thefoodstuff marker is indicative of the manner in which the foodstuff wasobtained (grown, procured, caught, harvested, processed, cooked, etc.).In some embodiments, the foodstuff marker is indicative of thenutritional content of the foodstuff. In some embodiments, the foodstuffmarker is an allergen that may induce an allergic reaction if thefoodstuff from which the sample is obtained is consumed by a subject.

In some embodiments, the devices, systems and methods in the presentinvention further includes receiving or providing a report thatindicates the safety or harmfulness for a subject to consume the foodstuff from which the sample was obtained based on information includingthe measured level of the foodstuff marker. The information used toassess the safety of the foodstuff for consumption may include dataother than the type and measured amount of the foodstuff marker. Theseother data may include any health condition associated with the consumer(allergies, pregnancy, chronic or acute diseases, current prescriptionmedications, etc.).

The report may be generated by the device configured to read the CROFdevice, or may be generated at a remote location upon sending the dataincluding the measured amount of the foodstuff marker. In some cases, afood safety expert may be at the remote location or have access to thedata sent to the remote location, and may analyze or review the data togenerate the report. The food safety expert may be a scientist oradministrator at a governmental agency, such as the US Food and DrugAdministration (FDA) or the CDC, a research institution, such as auniversity, or a private company. In certain embodiments, the foodsafety expert may send to the user instructions or recommendations basedon the data transmitted by the device and/or analyzed at the remotelocation.

25 Blood Testing

Some exemplary embodiments of the application of the present inventionare in simple, rapid blood cell counting using a smartphone.

In some embodiments, the first plate and the second plate are selectedfrom a thin glass slide (e.g. 0.2 mm thick) or a thin plastic film (e.g.15 mm thick) of a relative flat surface, and each have an areas with alength and width in about 0.5 cm to 10 cm. The spacers are made ofglass, plastics, or other materials that would not deform significantlyunder a pressing. Before the sample deposition, the spacer are placed onthe first plate, the second plate or both; and the first plate, thesecond plate or both are optionally coated with reagent that facilitatethe blood counting (staining dyes and/or anticoagulant). The first plateand the second plate can be optionally sealed in a bag for easytransport and longer shelf life-time.

In blood cell count testing, only about 1 uL (microliter) (or about 0.1uL to 3 uL) of blood is needed for the sample, which can be taken from afinger or other human body location. The blood sample can be directlydeposited from human body (e.g. finger) onto the first plate and thesecond plate, without any dilution. Then the first plate and the secondplate are made facing each other, so that blood sample is between theinner surfaces of the first plate and the second plate. If the optionalreagents are pre-deposited (staining dyes or anticoagulant), they aredeposited on the inner surface for mixing with the sample. Then thefirst plate and the second plate are pressed by fingers or a simplemechanical device (e.g. a clip that presses using a spring). Under thepress, the inner spacing is reduced, the reduction will be eventuallystopped at the value set by the spacers' height and the final samplethickness is reached, which generally is equal to the final innerspacing. Since the final inner spacing is known, the final samplethickness become known, namely being quantified (measured) by thismethod.

If the blood sample is not diluted, after pressing (sample deformation)the spacers and hence the final sample thickness may be thin, e.g., less1 um, less 2 um, less 3 um, less 4 um, less 5 um, less 7 um, less 10 um,less 15 um, less 20 um, less 30 um, less 40 um, less 50 um, less 60 um,less 80 um, less 100 um, less 150 um, or any ranges between any of thetwo numbers. A thin final sample may be useful because if the finalsample thickness is thick, then many red cells may overlap during theimaging, which can make the cell counting inaccurate. For example, about4 um thick of whole blood without dilution will give about one layer ofblood red cells.

After the pressing, the sample may be imaged by a smartphone eitherdirectly or through an additional optical elements (e.g. lenses,filters, or light sources as needed). The image of the sample will beprocessed to identify the types of the cells as well as the cell number.The image processing can be done locally at the same smartphone thattakes the image or remotely but the final result transmitted back to thesmartphone (where the image is transmitted to a remote location and isprocessed there.) The smart phone will display the cell number for aparticular cell. In some cases, certain advices will be displayed. Theadvices can stored on the smartphone before the test or come from aremote machines or professionals.

In certain embodiments, reagents will be put on the inner surfaces ofthe first plate and/or the second plate using the methods and devicesdescribed in Section 5 (Reagent mixing).

A device or a method for the blood testing comprises (a) a device or amethod in paragraph described herein and (b) a plate spacing (i.e. thedistance between the inner surfaces of the two plates) at the closedconfiguration or a use of such spacing, wherein a undiluted whole bloodin the plate-spacing has an average inter-cell distance in the lateraldirection for the red blood cells (RBC) larger than the average diameterof the disk shape of the RBC.

A device or a method to arrange the orientation of a non-spherical cellcomprises (a) a device or a method in as described herein and (b) aplate spacing (i.e. the distance between the inner surfaces of the twoplates) at the closed configuration or a use of such spacing, whereinthe spacing less than the average size of the cell in its long direction(the long direction is the maximum dimension direction of a cell). Sucharrangement can improve the measurements of the sample volume (e.g. redblood cell volume).

In the present invention, the analytes in the blood tests includeprotein markers, a list of which may be found at the website of theAmerican Association for Clinical Chemistry).

26 Packages

Another aspect of the present invention is related to packaging, whichwould prolong the lifetime of the reagent used and facilitate the easyof the use.

In some embodiments, the plates in CROF with or without reagents are putinside a package, either one plate per package or more than one platesper package. In one embodiment, the first plate and second plate arepackaged in a different package before a use. In some embodiments,different assays share a common first plate or a common second plate.

In some embodiments, each of the packages is sealed. In someembodiments, the seal is for preventing the air, chemicals, moisture,contamination, or any combination of them from outside of the packagefrom entering inside the package. In some embodiments, the package isvacuum sealed or fill with nitrogen gas, or inner gases. In someembodiments, a material that can prolong a shelf-life-time of the plateand/or the reagents (including the capture agents, detection agents,etc.) is packaged inside the package with a plate.

In some embodiments, the package materials are a thin layer form, sothat the package can be easily torn apart by a human hand.

27 PoC, SmartPhone, and Network

An aspect of the invention is directed to a method for monitoring thehealth status of a subject, the method including: applying a sampleprovided from a subject to a CROF based detector configured to indicatean output that is representative of the sample; processing the detectoroutput with a device configured to acquire the detector output as inputdata and to analyze the input data to generate a report; and receivingthe report. The signal enhancing detector offers the advantages of fastdetection, simplified reader (e.g. replace large conventional reader bysmartphone), and lost cost.

Body Fluid. In certain embodiments, a sample may include various fluidor solid samples. In some instances, the sample can be a bodily fluidsample from the subject. In some instances, solid or semi-solid samplescan be provided. The sample can include tissues and/or cells collectedfrom the subject. The sample can be a biological sample. Examples ofbiological samples can include but are not limited to, blood, serum,plasma, a nasal swab, a nasopharyngeal wash, saliva, urine, gastricfluid, spinal fluid, tears, stool, mucus, sweat, earwax, oil, aglandular secretion, cerebral spinal fluid, tissue, semen, vaginalfluid, interstitial fluids derived from tumorous tissue, ocular fluids,spinal fluid, a throat swab, breath, hair, finger nails, skin, biopsy,placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavityfluids, sputum, pus, microbiota, meconium, breast milk and/or otherexcretions. The samples may include nasopharyngeal wash. Nasal swabs,throat swabs, stool samples, hair, finger nail, ear wax, breath, andother solid, semi-solid, or gaseous samples may be processed in anextraction buffer, e.g., for a fixed or variable amount of time, priorto their analysis. The extraction buffer or an aliquot thereof may thenbe processed similarly to other fluid samples if desired. Examples oftissue samples of the subject may include but are not limited to,connective tissue, muscle tissue, nervous tissue, epithelial tissue,cartilage, cancerous sample, or bone.

In certain embodiments, the subject may be a human or a non-humananimal. The subject may be a mammal, vertebrate, such as murines,simians, humans, farm animals, sport animals, or pets. In someembodiments, the subject may be a patient. In other embodiments, thesubject may be diagnosed with a disease, or the subject may not bediagnosed with a disease. In some embodiments, the subject may be ahealthy subject.

Device Reading

As summarized above, aspects of the method include processing the signalenhancing detector output with a device configured to acquire thedetector output as input data and process the input data to generate areport. Any device suitable for acquiring the detector output as inputdata and processing the input data to generate a report may be used. Insome embodiments, the device includes an optical recording apparatusthat is configured to acquire an optical detector output as input data.In certain instances, the optical recording apparatus is a camera, suchas a digital camera. The term “digital camera” denotes any camera thatincludes as its main component an image-taking apparatus provided withan image-taking lens system for forming an optical image, an imagesensor for converting the optical image into an electrical signal, andother components, examples of such cameras including digital stillcameras, digital movie cameras, and Web cameras (i.e., cameras that areconnected, either publicly or privately, to an apparatus connected to anetwork to permit exchange of images, including both those connecteddirectly to a network and those connected to a network by way of anapparatus, such as a personal computer, having an information processingcapability). In one example, the input data may include video imagingthat may capture changes over time. For example, a video may be acquiredto provide evaluation on dynamic changes in the sample.

In certain embodiments, the device acquires the detector output by meansof an adaptor that forms an interface between the device and thedetector. In certain embodiments, the interface is universal to becompatible with any device suitable for performing the subject method.Interfaces of interest include, but are not limited to, USB, firewire,Ethernet, etc. In certain embodiments, the device acquires the detectoroutput by wireless communication, including cellular, Bluetooth, WiFi,etc.

In certain embodiments, the device may have a video display. Videodisplays may include components upon which a display page may bedisplayed in a manner perceptible to a user, such as, for example, acomputer monitor, cathode ray tube, liquid crystal display, lightemitting diode display, touchpad or touchscreen display, and/or othermeans known in the art for emitting a visually perceptible output. Incertain embodiments, the device is equipped with a touch screen fordisplaying information, such as the input data acquired from thedetector and/or the report generated from the processed data, andallowing information to be entered by the subject.

In certain embodiments, the device is equipped with vibrationcapabilities as a way to alert the subject, for example, of a reportgenerated upon processing the detector output or in preparation foracquiring an output from the detector.

In certain embodiments, the device is configured to display informationon a video display or touchscreen display when a display page isinterpreted by software residing in memory of the device. The displaypages described herein may be created using any suitable softwarelanguage such as, for example, the hypertext mark up language (“HTML”),the dynamic hypertext mark up language (“DHTML”), the extensiblehypertext mark up language (“XHTML”), the extensible mark up language(“XML”), or another software language that may be used to create acomputer file displayable on a video or other display in a mannerperceivable by a user. Any computer readable media with logic, code,data, instructions, may be used to implement any software or steps ormethodology. Where a network comprises the Internet, a display page maycomprise a webpage of a suitable type.

A display page according to the invention may include embedded functionscomprising software programs stored on a memory device, such as, forexample, VBScript routines, JScript routines, JavaScript routines, Javaapplets, ActiveX components, ASP.NET, AJAX, Flash applets, Silverlightapplets, or AIR routines.

A display page may comprise well known features of graphical userinterface technology, such as, for example, frames, windows, scrollbars, buttons, icons, and hyperlinks, and well known features such as a“point and click” interface or a touchscreen interface. Pointing to andclicking on a graphical user interface button, icon, menu option, orhyperlink also is known as “selecting” the button, option, or hyperlinkA display page according to the invention also may incorporatemultimedia features, multi-touch, pixel sense, IR LED based surfaces,vision-based interactions with or without cameras.

In certain embodiments, the device may be configured to acquire datathat is not an output from the signal enhancing detector. Thus incertain cases, the device may be configured to acquire data that is notrepresentative of the sample provided by the subject but may still berepresentative of the subject. Such data include, but are not limited tothe age, sex, height, weight, individual and family medical history,etc. In certain embodiments, the device is configured to process theinput data acquired from the detector output combined with data that wasacquired independently of the detector output.

In certain embodiments the device may be configured to communicate overa network such as a local area network (LAN), wide area network (WAN)such as the Internet, personal area network, a telecommunicationsnetwork such as a telephone network, cell phone network, mobile network,a wireless network, a data-providing network, or any other type ofnetwork. In certain embodiments the device may be configured to utilizewireless technology, such as Bluetooth or RTM technology. In someembodiments, the device may be configured to utilize variouscommunication methods, such as a dial-up wired connection with a modem,a direct link such as TI, ISDN, or cable line. In some embodiments, awireless connection may be using exemplary wireless networks such ascellular, satellite, or pager networks, GPRS, or a local data transportsystem such as Ethernet or token ring over a LAN. In some embodiments,the device may communicate wirelessly using infrared communicationcomponents.

In certain embodiments, the device is configured to receive a computerfile, which can be stored in memory, transmitted from a server over anetwork. The device may receive tangible computer readable media, whichmay contain instructions, logic, data, or code that may be stored inpersistent or temporary memory of the device, or may somehow affect orinitiate action by the device. One or more devices may communicatecomputer files or links that may provide access to other computer files.

In some embodiments, the device is a personal computer, server, laptopcomputer, mobile device, tablet, mobile phone, cell phone, satellitephone, smartphone (e.g., iPhone, Android, Blackberry, Palm, Symbian,Windows), personal digital assistant, Bluetooth device, pager, land-linephone, or other network device. Such devices may becommunication-enabled devices. The term “mobile phone” as used hereinrefers to a telephone handset that can operate on a cellular network, aVoice-Over IP (VoIP) network such as Session Initiated Protocol (SIP),or a Wireless Local Area Network (WLAN) using an 802.11x protocol, orany combination thereof. In certain embodiments, the device can behand-held and compact so that it can fit into a consumer's wallet and/orpocket (e.g., pocket-sized).

In certain embodiments, the method includes transmitting thesample-derived data to a remote location where the transmitted data isanalyzed. The remote location may be a location that is different fromthe location where the device is located. The remote location mayinclude, but is not limited to, a hospital, doctor's office or othermedical facility, or a research laboratory. In some instances, theremote location may have a computer, e.g., a server, that is configuredto communicate with (i.e. receive information from and transmitinformation to) the device over a network. In some embodiments, thedevice may transmit data to a cloud computing infrastructure. The devicemay access the cloud computing infrastructure. In some embodiments,on-demand provision of computational resources (data, software) mayoccur via a computer network, rather than from a local computer. Thedevice may contain very little software or data (perhaps a minimaloperating system and web browser only), serving as a basic displayterminal connected to the Internet. Since the cloud may be theunderlying delivery mechanism, cloud-based applications and services maysupport any type of software application or service. Informationprovided by the device and/or accessed by the devices may be distributedover various computational resources. Alternatively, information may bestored in one or more fixed data storage unit or database.

In certain embodiments, the remote location includes a central databasestored in a data storage unit that receives and analyzes the datatransmitted from the device. The data storage units may be capable ofstoring computer readable media which may include code, logic, orinstructions for the processor to perform one or more step. In someembodiments, the received data is analyzed in a comparative fashion withdata contained in the central database and the result sent back to thesubject. Analyzing may include correcting raw data based on mathematicalmanipulation or correction and/or calibrations specific for the deviceor reagents used to examine the sample; calculation of a value, e.g., aconcentration value, comparison (e.g., with a baseline, threshold,standard curve, historical data, or data from other sensors), adetermination of whether or not a test is accurate, highlighting valuesor results that are outliers or may be a cause for concern (e.g., aboveor below a normal or acceptable range, or indicative of an abnormalcondition), or combinations of results which, together, may indicate thepresence of an abnormal condition, curve-fitting, use of data as thebasis of mathematical or other analytical reasoning (includingdeductive, inductive, Bayesian, or other reasoning), and other suitableforms of processing.

In certain embodiments, analyzing may involve comparing the analyzeddata with a database stored in a data storage unit at the remotelocation to retrieve instructions for a course of action to be performedby the subject. In some embodiments, the database may contain storedinformation that includes a threshold value for the analyte of interest.The threshold value may be useful for determining the presence orconcentration of the one or more analyte. The threshold value may beuseful for detecting situations where an alert may be useful. The datastorage unit may include any other information relating to samplepreparation or clinical tests that may be run on a sample. The datastorage unit may include records or other information that may be usefulfor generating a report relating to the analyzed data.

In certain embodiments, a health care professional is at the remotelocation. In other embodiments, a health care professional has access tothe data transmitted by the device at a third location that is differentfrom the remote location or the location of the device. A health careprofessional may include a person or entity that is associated with thehealth care system. A health care professional may be a medical healthcare provider. A health care professional may be a doctor. A health careprofessional may be an individual or an institution that providespreventive, curative, promotional or rehabilitative health care servicesin a systematic way to individuals, families and/or communities.Examples of health care professionals may include physicians (includinggeneral practitioners and specialists), dentists, physician assistants,nurses, midwives, pharmaconomists/pharmacists, dietitians, therapists,psychologists, chiropractors, clinical officers, physical therapists,phlebotomists, occupational therapists, optometrists, emergency medicaltechnicians, paramedics, medical laboratory technicians, medicalprosthetic technicians, radiographers, social workers, and a widevariety of other human resources trained to provide some type of healthcare service. A health care professional may or may not be certified towrite prescriptions. A health care professional may work in or beaffiliated with hospitals, health care centers and other servicedelivery points, or also in academic training, research andadministration. Some health care professionals may provide care andtreatment services for patients in private homes. Community healthworkers may work outside of formal health care institutions. Managers ofhealth care services, medical records and health information techniciansand other support workers may also be health care professionals oraffiliated with a health care provider.

In some embodiments, the health care professional may already befamiliar with the subject or have communicated with the subject. Thesubject may be a patient of the health care professional. In someinstances, the health care professional may have prescribed the subjectto undergo a clinical test. In one example, the health care professionalmay be the subject's primary care physician. The health careprofessional may be any type of physician for the subject (includinggeneral practitioners, and specialists).

Thus, a health care professional may analyze or review the datatransmitted from the device and/or the results of an analysis performedat a remote location. In certain embodiments, the health careprofessional may send to the subject instructions or recommendationsbased on the data transmitted by the device and/or analyzed at theremote location.

28 Control and Measure the Sample Thickness without Using Spacers

In some embodiments of the present invention, the spacers that are usedto regulate the sample or a relevant volume of the sample are replacedby (a) positioning sensors that can measure the plate inner spacing, and(b) the devices that can control the plate positions and move the platesinto a desired plate inner spacing based on the information provided thesensors. In some embodiment, all the spacers are replaced by translationstage, monitoring sensors and feedback system.

Measuring of Spacing and/or Sample Thickness Using Optical Method. Insome embodiments, the measuring (f) of the spacing between the innersurfaces comprises the use of optical interference. The opticalinterference can use multiple wavelength. For example, the light signaldue to the interference of a light reflected at the inner surface of thefirst plate and the second plate oscillate with the wavelength of thelight. From the oscillation, one can determine the spacing between theinner surfaces. To enhance the interference signal, one of the innersurfaces or both can be coated with light reflection material.

In some embodiments, the measuring (f) of the spacing between the innersurfaces comprises taking optical imaging (e.g. taking a 2D(two-dimensional)/3D (three-dimensional) image of the sample and theimage taking can be multiple times with different viewing angles,different wavelength, different phase, and/or different polarization)and image processing.

Measuring of Entire Sample Area or Volume Using Optical Methods. In someembodiments, the measuring (f) of the entire sample area or volumecomprises taking optical imaging (e.g. taking a 2D (two-dimensional)/3D(three-dimensional) image of the sample and the image taking can bemultiple times with different viewing angles, different wavelength,different phase, and/or different polarization) and image processing.The sample area means the area in the direction approximately parallelto the first plate and the second plate. The 3D imaging can use themethod of fringe projection profilometry (FPP), which is one of the mostprevalent methods for acquiring three-dimensional (3D) images ofobjects.

In some embodiments, the measuring of the sample area or volume byimaging comprises (a) calibration of the image scale by using a sampleof the known area or volume (e.g., The imager is a smartphone and thedimensions of the image taken by the phone can be calibrated bycomparing an image of the a sample of known dimension taken the samephone); (b) comparison of the image with the scale markers (rulers)placed on or near the first plate and second plate (discussed furtherherein), and (c) a combination of thereof.

As used herein, light may include visible light, ultraviolet light,infrared light, and/or near infrared light. Light may includewavelengths in the range from 20 nm to 20,000 nm.

29 Other Descriptions of Embodiments

The following methods, devices and systems are provided. Theseembodiments may be implemented using any of the components, materials,parameters or steps described above or below. The following embodimentsuse a CROF plate.

Embodiment 1. A method for analyzing a liquid sample, comprising:

-   -   (a) obtaining a sample that contains an analyte;    -   (b) obtaining a first and second plates that are movable        relative to each other into different configurations, wherein        each plate has a sample contact surface that is substantially        planar, one or both plates are flexible, and one or both of the        plates comprise spacers that are fixed with a respective sample        contacting surface, and wherein the spacers have a predetermined        substantially uniform height and a predetermined constant        inter-spacer distance that is at least about 2 times larger than        the size of the analyte, up to 200 um (micrometer);    -   (c) depositing the sample on one or both of the plates when the        plates are configured in an open configuration, wherein the open        configuration is a configuration in which the two plates are        either partially or completely separated apart and the spacing        between the plates is not regulated by the spacers;    -   (d), after (c), using the two plates to compress at least part        of the sample into a layer of substantially uniform thickness        that is confined by the sample contact surfaces of the plates,        wherein the uniform thickness of the layer is regulated by the        spacers and the plates, wherein the compressing comprises:        -   bringing the two plates together; and        -   conformable pressing, either in parallel or sequentially, an            area of at least one of the plates to press the plates            together to a closed configuration, wherein the conformable            pressing generates a substantially uniform pressure on the            plates over the at least part of the sample, and the            pressing spreads the at least part of the sample laterally            between the sample contact surfaces of the plates, and            wherein the closed configuration is a configuration in which            the spacing between the plates in the layer of uniform            thickness region is regulated by the spacers; and    -   (e) analyzing the analyte in the layer of uniform thickness        while the plates are the closed configuration;

wherein a conformable pressing is a method that makes the pressureapplied over an area is substantially constant regardless the shapevariation of the outer surfaces of the plates; and

wherein the parallel pressing applies the pressures on the intended areaat the same time, and a sequential pressing applies the pressure on apart of the intended area and gradually move to other area.

Embodiment 2. A device for analyzing a liquid sample, comprising:

a first plate and a second plate, wherein:

i. the plates are movable relative to each other into differentconfigurations;

ii. one or both plates are flexible;

iii. each of the plates has, on its respective surface, a sample contactarea for contacting a sample that contains an analyte,

iv. one or both of the plates comprise spacers that are fixed with arespective sample contact area, wherein the spacers have a predeterminedsubstantially uniform height and a predetermined constant inter-spacerdistance that is at least about 2 times larger than the size of theanalyte, up to 200 um, and wherein at least one of the spacers is insidethe sample contact area;

wherein one of the configurations is an open configuration, in which:the two plates are separated apart, the spacing between the plates isnot regulated by the spacers, and the sample is deposited on one or bothof the plates; and

wherein another of the configurations is a closed configuration which isconfigured after the sample deposition in the open configuration; and inthe closed configuration: at least part of the sample is compressed bythe two plates into a layer of highly uniform thickness, wherein theuniform thickness of the layer is confined by the sample contactsurfaces of the plates and is regulated by the plates and the spacers.

Embodiment 3. A method for analyzing a blood sample, comprising:

-   -   (a) obtaining a blood sample;    -   (b) obtaining a first and second plates that are movable        relative to each other into different configurations, wherein        each plate has a sample contact surface that is substantially        planar, one or both plates are flexible, and one or both of the        plates comprise spacers that are fixed with a respective sample        contacting surface, and wherein the spacers have:

i. a predetermined substantially uniform height,

ii. a shape of pillar with substantially uniform cross-section and aflat top surface;

iii. a ratio of the width to the height equal or larger than one;

iv. a predetermined constant inter-spacer distance that is in the rangeof 10 □m to 200 □m;

v. a filling factor of equal to 1% or larger; and

vi. a product of the filling factor and the Young's modulus of thespacer is 2 MPa or larger; and

-   -   (c) depositing the blood sample on one or both of the plates        when the plates are configured in an open configuration, wherein        the open configuration is a configuration in which the two        plates are either partially or completely separated apart and        the spacing between the plates is not regulated by the spacers;

(d), after (c), using the two plates to compress at least part of theblood sample into a layer of substantially uniform thickness that isconfined by the sample contact surfaces of the plates, wherein theuniform thickness of the layer is regulated by the spacers and theplates, and has an average value in the range of 1.8 □m to 3 □m with avariation of less than 10%, wherein the compressing comprises:

-   -   bringing the two plates together; and    -   conformable pressing, either in parallel or sequentially, an        area of at least one of the plates to press the plates together        to a closed configuration, wherein the conformable pressing        generates a substantially uniform pressure on the plates over        the at least part of the sample, and the pressing spreads the at        least part of the sample laterally between the sample contact        surfaces of the plates, and wherein the closed configuration is        a configuration in which the spacing between the plates in the        layer of uniform thickness region is regulated by the spacers;        and    -   (e) analyzing the blood in the layer of uniform thickness while        the plates are the closed configuration;

wherein the filling factor is the ratio of the spacer contact area tothe total plate area;

wherein a conformable pressing is a method that makes the pressureapplied over an area is substantially constant regardless the shapevariation of the outer surfaces of the plates; and

wherein the parallel pressing applies the pressures on the intended areaat the same time, and a sequential pressing applies the pressure on apart of the intended area and gradually move to other area.

Embodiment 4. A device for analyzing a liquid sample, comprising:

a first plate and a second plate, wherein:

v. the plates are movable relative to each other into differentconfigurations;

vi. one or both plates are flexible;

vii. each of the plates has, on its respective surface, a sample contactarea for contacting a blood sample;

viii. one or both of the plates comprise spacers that are fixed with arespective plate, wherein the spacers have a predetermined substantiallyuniform height and a predetermined constant inter-spacer distance thatis in the range of 7 □m to 200 □m and wherein at least one of thespacers is inside the sample contact area;

wherein one of the configurations is an open configuration, in which:the two plates are separated apart, the spacing between the plates isnot regulated by the spacers, and the sample is deposited on one or bothof the plates; and

wherein another of the configurations is a closed configuration which isconfigured after the sample deposition in the open configuration; and inthe closed configuration: at least part of the sample is compressed bythe two plates into a layer of highly uniform thickness, wherein theuniform thickness of the layer is confined by the inner surfaces of thetwo plates and is regulated by the plates and the spacers, and has anaverage value in the range of 1.8 □m to 3 □m with a small variation.

Embodiment 5. A method for locally binding a target entity in a portionof a liquid sample, comprising:

-   -   (a) obtaining a sample that contains a target entity that is        capable of diffusing in the sample;    -   (b) obtaining a first and second plates that are movable        relative to each other into different configurations, wherein        one or both of the plates comprise spacers that are fixed on a        respective plate, wherein the spacers have a predetermined        substantially uniform height, and wherein the first plate        comprises, on its surface, a binding site that has a        predetermined area and binds to and immobilizes the target        entity;    -   (c) depositing the sample on one or both of the plates when the        plates are configured in an open configuration, wherein the open        configuration is a configuration in which the two plates are        either partially or completely separated apart and the spacing        between the plates is not regulated by the spacers;    -   (d) after (c), compressing the sample by bringing the two plates        into a closed configuration, wherein the closed configuration is        a configuration in which at least part of the sample is        compressed into a layer of uniform thickness that is in touch to        and confined by the inner surfaces of the two plates and that in        touch to the binding site, wherein the uniform thickness of the        layer is regulated by the spacers and the plates, is less than        250 um, and is substantially less than the linear dimension of        the predetermined area of the binding site;

(e) after (d) and while the plates are in the closed configuration,either:

(1) incubating the sample for a relevant time length and then stoppingthe incubation; or

(2) incubating the sample for a time that is equal or longer than theminimum of a relevant time length, and then assessing, within a timeperiod that is equal or less than the maximum of the relevant length oftime, the binding of target entity to in the binding site;

wherein the relevant time length is:

i. equal to or longer than the time that it takes for the target entityto diffuse across the thickness of the uniform thickness layer at theclosed configuration; and

ii. significantly shorter than the time that it takes the target entityto laterally diffuse across the minimum lateral dimension of the bindingsite;

wherein at the end of the incubation in (1) or during the assessing in(2), the majority of the target entity bound to the binding site is froma relevant volume of the sample;

wherein the incubation allows the target entity to bind to the bindingsite, and wherein the relevant volume is a portion of the sample that isabove the binding site at the closed configuration.

Embodiment 6. A device for locally binding target entity in a portion ofa liquid sample, comprising:

a first plate and a second plate, wherein:

-   -   i. the plates are movable relative to each other into different        configurations; one or both plates are flexible;

iii. each of the plates has, on its respective surface, a sample contactarea for contacting a sample that contains an entity which is capable ofdiffusing in the sample,

iv. one of the plates has, on its sample contact area, a binding sitethat has a predetermined area and binds and immobilize the targetentity;

v. one or both of the plates comprise spacers that are fixed with arespective plate, wherein the spacers have a predetermined substantiallyuniform height and a predetermined constant inter-spacer distance, andwherein at least one of the spacers is inside the sample contact area;

wherein one of the configurations is an open configuration, in which:the two plates are either partially or completely separated apart, thespacing between the plates is not regulated by the spacers, and thesample is deposited on one or both of the plates, and

wherein another of the configurations is a closed configuration which isconfigured after the sample deposition in the open configuration; and inthe closed configuration: at least part of the sample is compressed bythe two plates into a layer of uniform thickness, wherein at least apart of the uniform thickness layer is over the binding site, andwherein the uniform thickness of the layer is confined by the innersurfaces of the two plates, is regulated by the plates and the spacers,is less than 250 um, and is substantially less than the average lineardimension of the predetermined area of the binding site.

Embodiment 7. A method for locally releasing a reagent into a portion ofa liquid sample, comprising:

-   -   (a) obtaining a sample;    -   (b) obtaining a first and second plates that are movable        relative to each other into different configurations, wherein:

(i) one or both of the plates comprise spacers that are fixed with arespective plate,

(ii) the spacers have a predetermined uniform height, and

(iii) the first plate comprises, on its surface, a storage site that hasa predetermined area and that comprises a reagent that, upon contactingthe sample, dissolves into the sample and diffuses in the sample;

-   -   (c) depositing the sample on one or both of the plates when the        plates are configured in an open configuration, wherein the open        configuration is a configuration in which the two plates are        either partially or completely separated apart and the spacing        between the plates is not regulated by the spacers;    -   (d) after (c), compressing the sample by bringing the two plates        into a closed configuration, wherein the closed configuration is        a configuration in which at least part of the sample is        compressed into a layer of uniform thickness that is confined by        the inner surfaces of the two plates and that covers the storage        site, wherein the uniform thickness of the layer is regulated by        the spacers and the plates, is less than 250 um, and is        substantially less than the linear dimension of the        predetermined area of the storage site;

(e) after (d) and while the plates are in the closed configuration,incubating the sample for a relevant time length and then stopping theincubation,

wherein the relevant time length is:

i. about equal to or longer than the time that it takes for the targetentity to diffuse across the thickness of the uniform thickness layer atthe closed configuration; and

ii. shorter than the time that it takes the target entity to laterallydiffuse across the linear dimension of the predetermined area of thebinding site;

thereby, after the incubation, the majority of the reagent thatinitially are on storage site are in the relevant volume of the sample,

wherein the incubation is a process to allow the reagent to bind or mixwith the sample, and wherein the relevant volume is a portion of thesample that is above the binding site at the closed configuration.

Embodiment 8. A device for locally releasing a reagent into a portion ofa liquid sample, comprising:

a first plate and a second plate, wherein:

-   -   i. the plates are movable relative to each other into different        configurations; ii. one or both plates are flexible;

vi. each of the plates has, on its respective surface, a sample contactarea for contacting a sample;

vii. one of the plates comprises, on its sample contact area, a storagesite that has a predetermined area and comprises an reagent that, uponcontacting the sample, dissolves into the sample, diffuses in thesample, and bind to the target entity;

viii. one or both of the plates comprise spacers that are fixed with arespective plate, wherein the spacers have (a) a predeterminedsubstantially uniform height that is 250 □m or less and is substantiallyless than the average linear dimension of the predetermined area of thereagent site, and (b) a predetermined constant inter-spacer distancethat is 200 □m or less, and wherein at least one of the spacers isinside the sample contact area;

wherein one of the configurations is an open configuration, in which:the two plates are either partially or completely separated apart, thespacing between the plates is not regulated by the spacers, and thesample is deposited on one or both of the plates, and

wherein another of the configurations is a closed configuration which isconfigured after the sample deposition in the open configuration; and inthe closed configuration: at least part of the sample is compressed bythe two plates into a layer of uniform thickness, wherein at least apart of the uniform thickness layer is over the binding site, andwherein the uniform thickness of the layer is confined by the innersurfaces of the two plates, is regulated by the plates and the spacers.

Embodiment 9. A method for reducing the time for binding a target entityin a relevant volume of a sample on a binding site on a plate surface,comprising:

-   -   (a) obtaining a sample that contains a target entity that is        capable of diffusing in the sample;    -   (b) obtaining a first and second plates that are movable        relative to each other into different configurations, wherein        one or both of the plates comprise spacers that are fixed on a        respective plate and one or both plates are flexible, wherein        the spacers have a substantially predetermined uniform height        and a predetermined constant inter-spacer distance, and wherein        the first plate comprises, on its surface, a binding site that        has a predetermined area and binds to and immobilizes the target        entity;    -   (c) depositing the sample on one or both of the plates when the        plates are configured in an open configuration, wherein the open        configuration is a configuration in which the two plates are        either partially or completely separated apart and the spacing        between the plates is not regulated by the spacers;    -   (d) after (c), compressing the sample by bringing the two plates        into a closed configuration, wherein the closed configuration is        a configuration in which the thickness of a relevant volume of        the sample is reduced, compared to that in the open        configuration of the plates, into a layer of substantially        uniform thickness having a lateral area of at least 1 mm2 that        is confined by the inner surfaces of the two plates and that        covers the binding site, wherein the uniform thickness of the        layer is regulated by the spacers and the plates, is less than        250 um, and is substantially less than the linear dimension of        the predetermined area of the binding site; wherein the relevant        volume is a portion or an entire volume of the sample;

wherein reducing the thickness of the relevant volume of the samplereduces the time for binding between the binding site and the targetentity in the relevant volume to reach equilibrium.

Embodiment 10. A device for locally binding target entity in a portionof a liquid sample, comprising:

a first plate and a second plate, wherein:

-   -   i. the plates are movable relative to each other into different        configurations; one or both plates are flexible;

iii. each of the plates has, on its respective surface, a sample contactarea for contacting a sample that contains an entity which is capable ofdiffusing in the sample,

iv. one of the plates has, on its sample contact area, a binding sitethat has a predetermined area and binds and immobilize the targetentity;

v. one or both of the plates comprise spacers that are fixed with arespective plate, wherein the spacers have a predetermined substantiallyuniform height and a predetermined constant inter-spacer distance, andwherein at least one of the spacers is inside the sample contact area;

wherein one of the configurations is an open configuration, in which:the two plates are either partially or completely separated apart, thespacing between the plates is not regulated by the spacers, and thesample is deposited on one or both of the plates, and

wherein another of the configurations is a closed configuration which isconfigured after the sample deposition in the open configuration; and inthe closed configuration: at least part of the sample is compressed bythe two plates into a layer of uniform thickness, wherein at least apart of the uniform thickness layer is over the binding site, andwherein the uniform thickness of the layer is confined by the innersurfaces of the two plates, is regulated by the plates and the spacers,is less than 250 um, and is substantially less than the average lineardimension of the predetermined area of the binding site; and

wherein reducing the thickness of the relevant volume of the samplereduces the time for binding between the binding site and the targetentity in the relevant volume to reach equilibrium.

Embodiment 11. A method for parallel, multiplex, assaying of a liquidsample without fluidic isolation, comprising:

-   -   (a) obtaining a sample that contains one or more target        analytes, which are capable of diffusing in the sample;        -   (b) obtaining a first and second plates that are movable            relative to each other into different configurations,            wherein:

i. one or both of the plates comprise spacers that are fixed with arespective plate and one or both plates are flexible,

ii. the spacers have a predetermined substantially uniform height and apredetermined constant inter-spacer distance,

-   -    iii. the first plate has, on its surface, one or a plurality of        binding sites that each has a predetermined area comprising a        capture agent that binds and immobilizes a corresponding target        analyte of (a); and        -   iv. the second plate has, on its surface, one or a plurality            of corresponding storage sites that each has a predetermined            area and comprises a detection agent of a concentration            that, upon contacting the sample, dissolves into the sample            and diffuses in the sample,        -   wherein each capture agent, target analyte and corresponding            detection agent is capable of forming a capture agent-target            analyte-detection agent sandwich in a binding site of the            first plate;    -   (c) depositing the sample on one or both of the plates when the        plates are configured in an open configuration, wherein the open        configuration is a configuration in which the two plates are        either partially or completely separated apart and the spacing        between the plates is not regulated by the spacers;

(d) after (c), compressing the sample by bringing the two plates into aclosed configuration, wherein the closed configuration is aconfiguration in which:

i. at least part of the sample is compressed into a layer of uniformthickness that is in contact with and confined by the inner surfaces ofthe two plates and that is in contact with the one or a plurality ofbinding sites and the one or a plurality of storage sites,

ii the one or a plurality of corresponding storage sites are over theone or a plurality of binding sites, and

iii. the uniform thickness of the layer is regulated by the spacers andthe plates, is less than 250 um, and is substantially less than thelinear dimension of the predetermined area of each storage site;

(e) after (d) and while the plates are in the closed configuration,either:

(1) incubating the sample for a relevant time length and then stoppingthe incubation; or

(2) incubating the sample for a time that is equal or longer than theminimum of a relevant time length and then assessing, within a timeperiod that is equal or less than the maximum of the relevant length oftime, the binding of each target analyte to a binding site;

wherein the relevant time length is:

i. equal to or longer than the time that it takes for a target analyteof (a) to diffuse across the thickness of the uniform thickness layer atthe closed configuration; and

ii. significantly shorter than the time that it takes a target analyteof (a) to laterally diffuse across the smallest linear dimension of thepredetermined area of a storage site or binding site;

thereby producing a reaction in which, at the end of the incubation in(1) or during the assessing in (2), the majority of the captureagent-target analyte-detection agent sandwich bound to each binding siteis from a corresponding relevant volume of the sample;

wherein the incubation allows each target analyte to bind to a bindingsite and a detection agent, wherein the corresponding relevant volume isa portion of the sample that is above the corresponding storage site atthe closed configuration, wherein the separation between the edges ofthe neighboring storage sites and the separation between the edges ofthe neighboring binding sites are larger than the distance that a targetanalyte or detection agent can diffuse in the relevant time, and whereinthere is no fluidic isolation between the binding site sites and/or thestorage sites.

Embodiment 12. A device for parallel, multiplex, assaying of a liquidsample without fluidic isolation, comprising a first plate and a secondplate, wherein:

i. the plates are movable relative to each other into differentconfigurations; one or both plates are flexible;

ii. one or both of the plates comprise spacers that are fixed with arespective plate; and the spacers have a predetermined substantiallyuniform height and a predetermined constant inter-spacer distance;

iii. each of the plates has, on its respective surface, a sample contactarea for contacting a sample that contains a sample that contains one ormore target analytes which is capable of diffusing in the sample,

iv. the first plate has, on its surface, one or a plurality of bindingsites that each has a predetermined area comprising a capture agent thatbinds and immobilizes a corresponding target analyte of the sample; and

v. the second plate has, on its surface, one or a plurality ofcorresponding storage sites that each has a predetermined area andcomprises a detection agent of a concentration that, upon contacting thesample, dissolves into the sample and diffuses in the sample,

wherein each capture agent, target analyte and corresponding detectionagent is capable of forming a capture agent-target analyte-detectionagent sandwich in a binding site of the first plate;

wherein one of the configurations is an open configuration, in which:the two plates are either partially or completely separated apart, thespacing between the plates is not regulated by the spacers, and thesample is deposited on one or both of the plates, and

wherein another of the configurations is a closed configuration which isconfigured after the sample deposition in the open configuration; and inthe closed configuration:

i. at least part of the sample is compressed into a layer of uniformthickness that is in contact with and confined by the inner surfaces ofthe two plates and that covers the one or a plurality of binding sitesand the one or a plurality of storage sites,

ii the one or a plurality of corresponding storage sites are over theone or a plurality of binding sites, and

iii. the uniform thickness of the layer is regulated by the spacers andthe plates, is less than 250 um, and is substantially less than thelinear dimension of the predetermined area of each storage site; and

iv. there is no fluidic isolation between the binding site and/or thestorage sites.

wherein the separation between the edges of the neighboring storagesites and the separation between the edges of the neighboring bindingsites are larger than the distance that a target analyte or detectionagent can diffuse in the relevant time, and wherein there is no fluidicisolation between the binding site sites and/or the storage sites.

Embodiment 13A. A system for rapidly analyzing a sample using a mobilephone comprising:

-   -   (a) a CROF device, wherein one or both plates of the CROF device        are movable relative to each other into different        configurations; wherein:

i. one of the configurations is an open configuration, in which: the twoplates are either partially or completely separated apart, the spacingbetween the plates is not regulated by the spacers, and the sample isdeposited on one or both of the plates, and

ii. another of the configurations is a closed configuration which isconfigured after the sample deposition in the open configuration; and inthe closed configuration: at least part of the sample is compressed bythe two plates into a layer of uniform thickness, and wherein theuniform thickness of the layer is in touch with and confined by theinner surfaces of the two plates, is regulated by the plates and thespacers;

-   -   (b) a mobile communication device comprising:

i. one or a plurality of cameras for the detecting and/or imaging thesample;

ii. electronics, signal processors, hardware and software for receivingand/or processing the detected signal and/or the image of the sample andfor remote communication; and

-   -   (c) a light source from either the mobile communication device        or an external source.

Embodiment 13B. A method for rapidly analyzing a sample using a mobilephone, comprising:

(a) depositing a sample on the CROF device of a system of Embodiment13A;

(b) assaying the sample deposited on the CROF device to generate aresult; and

(c) communicating the result from the mobile communication device to alocation remote from the mobile communication device.

Embodiment 14. A method for analyzing a liquid sample, comprising:

-   -   (a) obtaining a sample that contains an analyte that is capable        of diffusing in the sample;    -   (b) obtaining a first and second plates that are movable        relative to each other into different configurations, wherein        one or both of the plates comprise spacers that are fixed with a        respective plate, wherein the spacers have a predetermined        uniform height, and wherein the first plate comprises, on its        surface, an analyte assay area that has a predetermined area;    -   (c) depositing the sample on one or both of the plates when the        plates are configured in an open configuration, wherein the open        configuration is a configuration in which the two plates are        either partially or completely separated apart and the spacing        between the plates is not regulated by the spacers;    -   (d), after (c), using the two plates to compress at least part        of the sample into a layer of uniform thickness that is confined        by the inner surfaces of the two plates, wherein at least a part        of the layer is over the analyte assay area, wherein the uniform        thickness of the layer is regulated by the spacers and the        plates, and is substantially less than the linear dimension of        the predetermined lateral area of the analyte assay area,        wherein the compressing comprises:        -   bringing the two plates together; and        -   applying an external force on the outer surfaces of the            plates to press the plates together to a closed            configuration, wherein the force generates pressure on the            plates over the at least part of the sample, and the            pressing spreads the at least part of the sample laterally            between the inner surfaces of the plates, and wherein the            closed configuration is a configuration in which the spacing            between the plates in the layer of uniform thickness region            is regulated by the spacers;        -   (e) incubating the sample while the plates are in the closed            configuration, for a time that is: (i) about equal or longer            then the time that it takes the analyte to diffuse across            the thickness of the uniform thickness layer, and (i)            significantly shorter than the time that it takes the            analyte to diffuse across the area of the analyte assay            area; and    -   (f) immediately after (e) either stopping the incubation and        measuring

the analyte in the assay area, or continuing the incubation while theplates are the closed configuration and measuring the analyte in theassay area in a time that is significantly shorter than the time that ittakes the analyte to diffuse across the area of the analyte assay area.

The following descriptions may be applied to embodiments 1-14, as setforth above.

In any embodiment that uses CROF, the spacers can be inside of thesample area and inside the relevant area of the sample for gooduniformity of the sample thickness control.

In any embodiment that uses CROF, at least one of two plate can beplastic film of a thickness from 1 um to 50 um.

In any embodiment that uses CROF, at least one of two plate can beplastic film of a thickness from 50 um to 100 um.

In any embodiment that uses CROF, at least one of two plate can beplastic film of a thickness from 100 um to 150 um.

In any embodiment that uses CROF, at least one of two plate can beplastic film of a thickness from 150 um to 250 um.

In any embodiment that uses CROF, both two plates can be a plastic filmof a thickness that each of them is independently selected from 10 um to300 um.

In any embodiment that uses CROF, both two plates can be a plastic filmof a thickness that each of them is independently selected from 100 umto 200 um.

In any embodiment that uses CROF, both two plates can be a plastic filmof a thickness that each of them is independently selected from 10 um to100 um.

In any embodiment that uses CROF, the height of the spacer on the platecan be in the range of 5 nm to 100 nm.

In any embodiment that uses CROF, the height of the spacer on the platecan be in the range of 100 nm to 500 nm

In any embodiment that uses CROF, the height of the spacer on the platecan be in the range of 500 nm to 1 um

In any embodiment that uses CROF, the height of the spacer on the platecan be in the range of 1 to 2 um

In any embodiment that uses CROF, the height of the spacer on the platecan be in the range of 2 to 5 um.

In any embodiment that uses CROF, the height of the spacer on the platecan be in the range of 5 to 10 um.

In any embodiment that uses CROF, the height of the spacer on the platecan be in the range of 10 to 30 um.

In any embodiment that uses CROF, the height of the spacer on the platecan be in the range of 30 to 50 um.

In any embodiment that uses CROF, the height of the spacer on the platecan be in the range of 50 to 100 um.

In any embodiment that uses CROF, the inter spacer distance (IDS) is nogreater than 200 um.

In any embodiment that uses CROF, the inter spacer distance (IDS) is nogreater than 150 um.

In any embodiment that uses CROF, the inter spacer distance (IDS) is nogreater than 100 um.

In any embodiment that uses CROF, the inter spacer distance (IDS) is nogreater than 80 um, e.g., no greater than 60 um, no greater than 40 um,or no greater than 20 um.

In any embodiment that uses CROF, the width to height ratio of thespacers is at least 1.5 (e.g., at least 2, at least 3, at least 4 or atleast 5).

In any embodiment that uses CROF, the ratio of pillar width to pillarheight can be at least 1, at least 2, at least 5, or at least 10.

In any embodiment that uses CROF, the distance between the plates may bein the range of 2-50 um and any assay may have a saturation time of lessthen 1 minute.

In any embodiment that uses CROF, the method includes a wash.

In any embodiment that uses CROF, the method does not include a wash.

In any embodiment that uses CROF, the method has a sensitivity of lessthan 1 nM, e.g., 0.1 nmol, 10 pmol, 1 pmol, 0.1 pmol, 10 fmole, 1 fmoleor 0.1 fmol, after an incubation of less then 1 minute.

In any embodiment that uses CROF, the ratio of the period to the spacerwidth may be less than about 7.0 (e.g., about 7.0 to 1.0), particularlywhen the pillar height is less than about 100 um.

In any embodiment that uses CROF, a plate may have a thickness of 20-200um, e.g., 10-50 or 50-200 um.

In any embodiment that uses CROF, the sample volume may be less than 0.5um, e.g., less than 0.5 um, less than 0.4 um, less than 0.3 um, lessthan 0.2 um, or less than 0.1 um.

In any embodiment that uses CROF, the interspacing distance may be lessthan 200 um, e.g., 20-200 um, 20-50 um or 50-200 um.

Other embodiments. In a preferred embodiment for reducing a saturationincubation time of a binding process, a regent mixing process, acombination of two, or other process, the final sample thickness at aclosed configuration is less than 0.5 um (micron). In another preferredembodiment, the final sample thickness is in a range of 0.5 um to 1 um.In another preferred embodiment, the final sample thickness is in arange of 1 um to 4 um. In another preferred embodiment, the final samplethickness is in a range of 4 um to 10 um. In another preferredembodiment, the final sample thickness is in a range of 10 um to 30 um.In another preferred embodiment, the final sample thickness is in arange of 30 um to 100 um.

In a preferred embodiment for reducing a saturation incubation time of abinding process, a regent mixing process, a combination of two, or otherprocess, the final sample thickness is selected to make the saturationincubation time less than 2 sec.

In another preferred embodiments, the final sample thickness is selectedto make the saturation incubation time in a range of less than 4 sec,less than 8 sec, less than 12 sec, less than 20 sec, less than 30 sec,less than 40 sec, less than 60 sec, less than 120 sec, less than 300sec, less than 420 sec, or a range between any two of the values.

In any embodiment that uses CROF, the device may be compressed by handfor a period of less than 1 minute, e.g., less than 10 sec.

In certain embodiments, the CROF device is integrated a microfluidicplatform or device. The microfluidic device may be configured to havedifferent areas for receiving a sample, detecting analytes in the samplewith a CROF device, collecting waste material in a reservoir, etc. Thus,in certain embodiments, the microfluidic channel platform may includefluid handling components to direct a sample applied to a samplereceiving area of the microfluidic device to a CROF device configured todetect an analyte, as described above. The fluid handling components maybe configured to direct one or more fluids through the microfluidicdevice. In some instances, the fluid handling components are configuredto direct fluids, such as, but not limited to, a sample solution,buffers and the like. Liquid handling components may include, but arenot limited to, passive pumps and microfluidic channels. In some cases,the passive pumps are configured for capillary action-drivenmicrofluidic handling and routing of fluids through the microfluidicdevice disclosed herein. In certain instances, the microfluidic fluidhandling components are configured to deliver small volumes of fluid,such as 1 mL or less, such as 500 pL or less, including 100 pL or less,for example 50 pL or less, or 25 pL or less, or 10 pL or less, or 5 pLor less, or 1 pL or less. Thus, in certain embodiments, no externalsource of power is required to operate the microfluidic device andperform the devices, systems and methods in the present invention.

In certain embodiments, the microfluidic device has dimensions in therange of 5 mm×5 mm to 100 mm×100 mm, including dimensions of 50 mm×50 mmor less, for instance 25 mm×25 mm or less, or 10 mm×10 mm or less. Incertain embodiments, the microfluidic device has a thickness in therange of 5 mm to 0.1 mm, such as 3 mm to 0.2 mm, including 2 mm to 0.3mm, or 1 mm to 0.4 mm.

In certain embodiments, the CROF device is disposed within a container,e.g., a well of a multi-well plate. The CROF device also can beintegrated into the bottom or the wall of a well of a multi-well plate.

In some embodiments, a support containing a CROF device, such as amicrofluidic device or multi-well plate, may have an identifier for theCROF device that is contained in the support. An identifier may be aphysical object formed on the support, such as a microfluidic device.For example, the identifier may be read by a handheld device, such as amobile phone or a smart phone, as described above.

In some embodiments, a camera may capture an image of the identifier andthe image may be analyzed to identify the CROF device contained in themicrofluidic device. In one example, the identifier may be a barcode. Abarcode may be a 1D or 2D barcode. In some embodiments, the identifiermay emit one or more signal that may identify the signal enhancingdetector. For example, the identifier may provide an infrared,ultrasonic, optical, audio, electrical, or other signal that mayindicate the identity of the CROF device. The identifier may utilize aradiofrequency identification (RFID) tag.

The identifier may contain information that allows determination of thespecific type of CROF device present in a microfluidic device ormulti-well plate. In certain embodiments, the identifier provides a keyto a database that associates each identifier key to informationspecific to the type of CROF device present in a microfluidic device ormulti-well plate. The information specific to the type of CROF devicemay include, but are not limited to, the identity of the analytes whichthe CROF device configured to detect, the coordinates of the positionwhere a specific analyte may bind on the CROF device, the sensitivity ofdetection for each analyte, etc. The database may contain otherinformation relevant to a specific CROF device, including an expirationdate, lot number, etc. The database may be present on a handheld device,provided on a computer-readable medium, or may be on a remote serveraccessible by a handheld device.

In certain embodiments, when the CROF card (e.g. CROF plate) in theclosed configure ration, the total thickness of the CROF card is therange of 10 um to 3 mm (e.g. in the range of 10 um to 100 um, 100 to 500um, 500 um to 1 mm, 1 mm to 2 mm or 2 mm to 3 mm); and the lateraldimension of CROF card is in the range of 2 mm to 50 mm (e.g. 2 mm to 5mm, 5 mm to 10 mm, 10 mm to 20 mm, 20 mm to 30 mm, 30 mm to 40 mm or 40mm to 50 mm), wherein the x and y direction takes respectively a valuein the range.

In certain embodiments, the CROF plate slide in and out the opticaladaptor for a testing.

In certain embodiment, the optical adaptor has a thickness in the rangeof 2 mm to 40 mm (e.g. 2 to 5 mm, 5 to 10 mm, 10 to 20 mm, 20 to 30 mm,or 30 to 40 mm) and a lateral dimension in the range of 10 mm to 100 mm(e.g. 10 to 20 mm, 20 to 30 mm, 30 to 40 mm, 40 to 50 mm, 50 to 60 mm,50 to 60 mm, 60 to 70 mm, 70 to 80 mm, or 80 to 100 mm), wherein aparticular thickness, x-lateral dimension and y lateral dimension takesone of the value in the range, respectively

In certain embodiments, the spacers for testing white blood cells is therange of 2 um to 40 um (e.g. 2 to 10 um, 10 to 20 um, 20 to 30 um, or 30um to 40 um).

30. Homogenous Assay Using a Signal Amplification Surface

In many applications of an assay, particularly in PoC or other fastassays, it is desirable to avoid washing steps. One aspect of thepresent invention is related to the devices, systems, and methods thatcan avoid washing of the assay.

By incorporating and/or using a signal amplification surface, thedisclosed devices, systems, and methods may facilitate performing assayswithout washing. The surface amplification surface may only amplify thelight emitted in a small distance from the surface (e.g. 20 nm, or 50nm, or 100 nm). One example of the surface amplification layer is D2PA.

31. An Example of Assay Acceleration Using CROF with a Ring (Enclosure)Spacer

An experiment has been performed for assay acceleration that uses apolystyrene thin film as one of the CROF plate, a thin glass as theother plate, and a wax ring was the spacer, and was fixed on thepolystyrene plate. During a CROF process, 2 uL (microliter) of samplewas dropped inside the ring spacer (and at the center, forming a smalldroplet as dropped) and was compressed by the two plates into a thinnerfilm with the spacing between the plates was regulated by the ringspacer (i.e. a closed configuration of two CROF plate). The plates werecompressed by hands. The sample thickness was found uniform at theclosed configuration of the plates. One main reason for the uniformityis that the volume of the sample is the same as the volume between thering spacers and two plates. Both immunoassay and DNA hybridizationassay were tested.

In the immunoassay testing (wax ring spacer of ˜40 um height and 0.8 cmdiameter), the Protein A was used as the capture agent and was coated onthe polystyrene surface, a labeled IgG was used as an analyte. Afterincubation for a binding between Protein A and the labeled IgG, theunbound IgG was washed away and the label of captured IgG was measured.Different incubation time were tested. Our experiment found that thebinding saturates in less than 1 min incubation time (i.e. after 1 minor less the signal of captured IgG will not change with the incubationtime). Such short saturation incubation time is expected for a 40 umspacing (hence sample thickness), since the diffusion time for IgG in asolution over a 40 um distance is about a few seconds.

We also tested the incubation of such direct assay in a normal 96wellplate with 3 mm thick sample thickness, and found that a typicalsaturation incubation time is about 120 min. If the incubation processis limited by diffusion of the labeled IgG, by reducing the samplethickness from 3 mm to 40 um reduced the incubation time from −120 minto 1.28 sec, which is consistent with our observation of sub-1 minsaturation incubation time.

In the DNA hybridization testing (wax ring spacer of ˜52 um height and0.7 cm diameter), the streptavidin-BSA was the molecular linking layeron the polystyrene substrate and was linked to biotinylated capturestrand, the capture strand captures labeled target strand throughhybridization. After an incubation, the un-hybridized target strand waswash away, and the label signal was tested. Different incubation timewere tested. Our experiment found that the hybridization saturates inwithin 30 sec incubation time (i.e. after 1 min or less the signal ofcaptured IgG will not change with the incubation time). Such shortsaturation incubation time is expected for a 52 um spacing (hence samplethickness), since the diffusion time for the target probe in a solutionover a 52 um distance is about a few seconds. (More details of theexperiments were disclosed in, e.g., provisional application Ser. No.62/202,989

As a references, the same assays with a thicker sample thickness weretested, we found that for 1 mm thick sample, it would require about 20mins to reach saturation incubation.

(More details of the experiments were disclosed in, e.g., provisionalapplication Ser. No. 62/202,989

32. Example of Assay Acceleration (QAX and QMAX) by CROF with PillarSpacers

E-1.1 QAX Assay with a CROFF Device of a Pillar Spacer Array of 30 UmSpacer Height, to Achieving an Saturation Incubation Time Less than 30Sec.

The QAX by CROF was tested and ˜30 sec saturation time was achieved. Theexperiment is illustrated in FIGS. 13 .a and b. In the experiment, thecapture agent and the labeled detection agent was predeposited and driedon one of the pair CROF plate before a CROF process, then the sample wasdropped on a plate and closed with another plate using a CROF process.The dropping the sample took a few seconds, the CROF process took lessthan 10 secs. Our experiment found that for 30 um spacer height, thesaturation incubation time is within 30 secs.

Plates, Samples, Reagents. (1) the CROF used the self-holding CROFdevice comprises (i) a 2.5 cm by 2.5 cm area X-Plate made of 175 umthick PMMA film with a spacer array in the sample contact area, wherethe spacer array has a rectangle lattice with a constant period of 120um/110 um (in x and y lateral direction respectively), all spacers arepillars and have the same of rectangle shape of the same spacer height30 um height and 40 um width in x and 30 um in y, and the spacers aremade of the sample material (PMMA) as the plate and are fabricated bynanoimprint the PMMA film with a mold (hence the spacers are fixed onthe plate with predetermined spacer height and inter spacer spacing of80 um); and (ii) a glass plate of planar surface (1 mm thick, 3 cm by 5cm). The surfaces of the X-Plate and glass plate are untreated and arehydrophilic for the sample. (2) The dry capture agent (cAb) of anti-IgGwere pre-coated on the glass plate before sample dropping and a CROFprocess; (3) The dry detection agent (dAb) of anti-IgG were pre-coatedon the X-Plate before sample dropping an da CROF process; and (4) Thesample is Human-IgG in BSA buffer with different conventionconcentration.Experimental steps and Results. A small volume of the sample with theanalytes (Human IgG) was dropped onto the surface of one of the platesof CROF devices described in E2-1. Initially the sample on the plateforms puddle, but by placing the other plate of the CROF device on thepaddle and compressing the two plates together, the original bloodpuddle spreads into a large-area sample film but ultra-thin (˜30 um)regulated by the spacer array, which are inside of the spread sample.Then, human hands uniformly pressed the X-Plate onto droplet (center tocenter) against the glass plate for 5-10 s, release the hand, wait 30 s,and the plates stay in their closed configuration.

Then different samples (with different CROF devices) were incubated indifferent times and washed and measured (optical signal). The resultsare in FIG. 13 .b, which shows that the saturation incubation time ofless than 30 secs for a QAX assay described in FIG. 13 .a.

E.1.2 QMAX Assay and Homogeneous Assay

QMAX has been tested experimentally using M-Plate (i.e. D2PA) for themagnification of the signal. Furthermore the QMAX assay was comparedwith QAX assay where is no M0Pate to magnify the signal. Bothheterogeneous (with wash) and homogenous (without wash) were tested. Thetest assay is human IgG fluorescence immunoassay using QAX & QMAX.

Materials and methods: X-Plate (30 um pillar height, 30 um×40 um pillarsize, 80 um ISD) 25 mm×25 mm; M-Plate, size 25 mm×25 mm; and the assayreagents (in coating order) were (a) DSU, Protein-A, anti-human IgG(coated and dry on the substrate plate), (b) human IgG (analyte), and(c) anti-human IgG-IR800 reagents (coated and dry on the storage site ofthe x-plate)

Results (also shown in FIG. 14 ): Our experiments showed that for a CROFdevice with 30 um spacing at a closed configuration, the saturationincubation is within 1 min, and the sensitivity for lumpsum reading isLoD=2 pM for QMAX with wash, LoD=10 pM for QMAX without wash(homogenous); LoD=200 pM for QAX with wash, and QAX without wash(homogenous) LoD=(cannot read, no difference for different analyteconcentration).

33. Additional Exemplary Experimental Testing and Preferred Embodiments

In this section, additional exemplary experimental testing andobservations, and additional preferred embodiments of the presentinvention are given, which were performed using the following conditionsand sharing the following common observations.

Volume of deposited sample. Unless specified otherwise, all the samplesdeposited on the plate of CROF have a unknown volume, namely, the exactvolume is not known at time of deposition.

Plates. In the CROF devices used this section, unless specifiedotherwise, one of the two plates, termed “X-Plate” is the only platethat has the spacers. The other plate, termed “the substrate plate”, hasa planar surface and does not have any spacers. Different materials(including glass, PMMA (polymethacrylate), and PS (polystyrene)) for theplate and the spacers, different plate thicknesses and spacer geometries(shapes and sizes) have been tested. The sample contact surface of eachplate is a planar surface (except the protruding spacers) with a surfacesmoothness variation typically less than 30 nm, but many of the planarsurfaces had surface flatness variation, which was caused by aflexibility of the plates, intrinsic surface flatness (not related tothe plate flexibility), or both. Some of the plates have an innersurface smoothness variation larger than 30 nm. The typical dimensionsof the plates used in the examples are, unless specified otherwise, atleast 25 mm wide and at least 25 mm long.

Spacers. Unless specified otherwise, all the spacers in the Section: (i)were fixed on the sample surface of the X-plate and fabricated byembossing the surface (hence the material of the spacers are the same asthe X-plate); (ii) were array of pillars that have a nearly uniformcross-section of a shape of rectangle or square with round corners, anearly straight sidewall with a tilt angle from the normal less than 5degree, a flat top surface, and uniform spacer height; and (iii) had afixed inner spacer distance (ISD) in each X and Y direction (note thespacing in X may be different from the spacing in Y) (See FIG. 17 .b).Furthermore, the lateral shape of the pillar spacers are either squareand rectangles with round corners; different spacer height, size,inter-spacer distance, shape, and materials were tested.

Fabrication of Spacers. The spacers embossed on the X-Plate surface werefabricated by nanoimprint, where a mold was pressed directly into theplate and embossed an originally completely flat surface into a flatsurface but having the pillar spacers protruding from the surface. Theembossing used a temperature higher than the glass transitiontemperature of the plastic material, where the plastic material can flowunder the embossing. The mold was fabricated by lithography and etching,and in some cases, by electroplating over a master mold. The mold wasmade in Si, silicon dioxide, or nickel.

FIG. 17 shows examples of the spacers fabricated on the plate. Thespacers were fabricated by direct imprinting of the plastic platesurface using a mold. FIGS. 17(a) and (b) is the top view of opticalmicrograph of a square spacer lattice. Top view of photograph of (a) 46um×46 um pillar spacer size and 54 um inter pillar distance, and (b) 10um×70 um pillar spacer size and 10 um pillar distance; and prospect viewSEM of (c) 30 um×40 um pillar spacer size of 2 um spacer height, and (d)30 um×40 um pillar spacer size of 30 um spacer height. The micrographsshow that (1) the top of the pillar spacer is very flat, (2) the spacerhas nearly uniform cross-section, and (3) the corners of the pillarspacer are round with a radius curvature about 1 um. A large radiuscurvature (e.g. less sharp edge) is preferred, since a sharp edge canlyse a cell or affect fluidic flow more than a rounded edge.

Using a surface profilometer, we measured the pillar height over 2 cm by2 cm area of the X-Plate. We found that the typical uniformity of thepillar spacer height of the X-Plate fabricated using the methodsdescribed above has an average variation of 4 nm, 10 nm, and 100 nm, anda relative average variation of 0.2%, 0.2% and 0.33%, respectively forthe spacer height of 2 um, 5 um, and 30 um.

Typical experiment procedure. As illustrate in FIG. 15 , first, a smallvolume (a few uL or less) of sample was deposited on either thesubstrate or the x-plate, which forms form a small paddle(s). Second theplates were brought together with overlaps of the sample surface of theplate. Third, hand is used to press the plates into a closedconfiguration, where the sample become a thin film with an area muchlarger than the original paddle. Fourth, the hand(s) was related. Andfifth, various measurements were performed at the closed configuration.Certain details of the steps are given below.

Sample deposition methods. Two sample deposition methods were used. Onemethod deposited a sample on the plates by a pipette. In another method,the blood samples were directly deposited from a subject finger (pickedby a tool) by making the blood on the subject and the plate in contact.There were not dilutions to the blood that were directly deposited fromthe finger to the plate. In our experiments, we found that the finalexperimental results is, unless specified, independent of the sampledeposition methods.

The samples depositions were performed inside a room and under astandard room conditions without any special temperature control or dustfilters. We found that under such conditions, the dusts fall on thesamples do not affect the final measurements results, because (1) theflexible plate used conformable to the dust, allowing the samplethickness in other areas still being regulated by the spacers and notaffecting the sample thickness self-holding, and (2) the area of havingdusts were only very small portion of the total available sample areaand the measurements were done the areas that were not affected by thedust. The selection of the non-dust area were done by optical imaging.

In some situations, the two plates have surface protection covers toreduce the number of dusts fall on the plates. In some situations, thetwo plates are placed together with sample surfaces inside to preventdusts and other contaminations.

Plate's surface wetting properties. We have measured the wettingproperties of different plate surfaces used in our exemplaryexperiments. The table below gives the measured contact angle of asample of 5 uL on a untreated or treated surfaces of different platematerials (glass, PMMA, and PS) and different surface geometry (flatsurface and X-plate sample surface) for different sample types (water,PBS buffer (Phosphate-Buffered Saline), and blood), where the X-plate is175 um thick PMMA, and its sample surface has an array of pillar (i.e.spacer) of 2 um height, 30 um×40 um lateral size, and 110 um/120 umperiod (i.e. 80 inter spacer distance).

Plate Material & Surface Water PBS Blood Untreated flat Glass 45° 46°46° Untreated flat PMMA 60° 57° 59° Untreated flat PS 61° 59° 58°Untreated X-Plate 62° 60° 58° (PMMA)

The experiments showed that (1) all untreated surfaces of glass, PMMA,and PS have a hydrophilic surface (i.e. the contact angle is less than90 degree); (2) the untreated glass surface has a smaller wetting angle(more hydrophilic) than untreated PMMA and PS; (3) the contact anglesare similar for water PBS and blood, and blood has slightly betterwetting than the water and PBS; (4) the untreated PMMA X-plate hasnearly the sample contact angle as the untreated PMMA plate; and (5) thesurface wetting properties can be, as expected, significantly altered bysurface treatment to become more hydrophilic or more hydrophobic.

Surface hydrophobicity of a plate can be changed by a surface treatment.For example, for PMMA X-plate, we made it more hydrophilic by exposed asurface in an oxygen plasma, and more hydrophobic treatment by treatingthe surface with tridecafluoro-1,1,2,2-tetrahydrooctyltrichlorosilane.The contact angles was 25, 27, 28 degree for hydrophobic treatedX-Plate, and 105, 104, and 103 degree for hydrophilic treated X-Plate,respectively for the samples of water, PBS buffer, and blood.

In the discussion below, unless particularly specified, all the samplesurface (i.e. the inner surface that contacts a sample) of the platesare untreated.

Area and height of deposited sample. We measured the sample area andheight on the plate when a water sample was deposited using a pipette onthe plates that were at an open configuration.

Liquid Untreated Untreated Volume Substrate: Glass PMMA 1 uL Diameter(mm): 2.4 2 Estimated Height 0.5 0.6 (mm): 2 uL Diameter (mm): 3.0 2.5Estimated Height 0.6 0.8 (mm): 5 uL Diameter (mm): 4.1 3.5 EstimatedHeight 0.9 1.0 (mm):

The experiments showed that a typical sample deposited on the plate atthe open configuration has a thickness much larger than that at theclosed configuration.

We observed that at a closed configuration of the plates, (1) the totalsample area expands from a few millimeter diameters to a few centimeter(depending upon the spacer height), and (2) if the spacer array has asquare lattice, then the area of the sample at the closed configurationof the plates is also nearly a square shape with the edge of the samplesquare aligned to the direction of the spacer square lattice. Therefore,it demonstrated that the final sample area at the closed configurationcan be controlled by using different spatial arrangements of thespacers. If the spacer has a rectangle lattice, then the final samplearea at the closed configuration should be rectangle. If the spacer is aradial circular pattern, then the final sample area at the closedconfiguration can be circular.

Hand-Press. In all the experiments in the Section 30, the plates in aCROF process were brought together and compressed into a closedconfiguration of the plates by human hand(s). In a final pressing foruniform sample thickness over a large area of the CROF plates, often athumb presses one area and rub into different areas of the CROF plates.A process that uses hand(s) to press a CROF device (plates) into aclosed configuration is referred as “hand-pressing”.

Self-Holding. We observed, unless stated otherwise, that after pressingthe CROF plates into the final configuration and releasing thecompressing force (e.g. the pressing hand), the thickness of the samplebetween the two plates was still regulated by the spacer height and waskept in a constant thickness for a long period time (until the sampleeventually dry). The observation is termed “self-holding”. Theself-holding is the capillary forces between the plates, the liquidsample, and the environment (e.g. air), caused by the surface tensions.We observed that the hand-pressing and self-holding of the CROF-devicegave excellent sample thickness, as illustrated in E-1.

Measurements for plate spacing and sample thickness. In all theexperiments below, the spacing between the inner surfaces (i.e. thesample surfaces) of the two plates at a closed configuration weremeasured by the Fabry-Pérot cavity resonance (FP resonance) caused bythe inner surfaces of the plates. Due to the optical index differencesbetween a sample (and air) and the inner surface, each inner surface ofthe plates acts as an optical reflector and the two inner surfaces forman optical cavity. The FP resonance spectra is periodic, and the innersurface spacing, h, (hence the sample thickness) at the opticalmeasurement point can be calculated from:

$h = \frac{c}{2n\Delta v}$

where c is the speed of light, Δv is the period in frequency domain, andn is the refractive index of the sample between the plates.

In our FP resonance testing, the light source had an area of aboutaround 2 um by 2 um. Typically, we measured the plate inner surfacespacing at 25 different points over 1.6 cm by 1.6 cm area round centerof the CROF-device, where the 25 points is 5×5 square lattice with aperiod (i.e. the distance between the two neighbor points) of 4 mm. Themeasurements stayed away from the regions that occupied by the spacers(i.e. pillars).

Since the inner surfaces and the sample are in contact at a closedconfiguration of the plates, the measured inner surface spacing is thesame as the sample thickness at the measurement point.

Average Sample Thickness, H. The average sample thickness, H, iscalculated using the plate spacing measured at the 25 points and theformula:

$H = {\frac{\sum_{i = 1}^{25}h_{i}}{25}.}$

Sample thickness deviation refers to the deviation of the sample averagethickness, H, over a given area from a predetermined spacer height, H₀:(H−H₀). And the relative sample thickness deviation is the deviationdivided by the predetermined spacer height: [(H−H₀)/H₀]. A positivethickness deviation means that the sample is in average thicker than thespacer height, and a negative thickness deviation means that the sampleis in average thinner than the spacer height.

Sample thickness uniformity. The uniformity of a sample thickness,

, over a given area is defined as the standard deviation of the samplethickness over the given area.

$\Delta = \sqrt{\frac{\sum_{i = 1}^{25}\left( {h_{i} - H} \right)^{2}}{25}}$

32.1 Sample Thickness Deviation and Uniformity in Hand-PressingSelf-Holding CROF

Experimentally, we studied the parameters in the CROF devices andprocess that can affect the sample thickness deviation and uniformity ata closed configuration of the plates after releasing the hands. We foundthat the parameters include, but not limited to, the inter-spacerdistance (IDS), spacer's shape and dimensions (e.g. spacer lateraldimension, spacer height, the ratio of the spacer width to the height,spacer area filling factors (the ratio of spacer area to the total areaor the ratio of spacer period to the width), the material mechanicalstrength (Young's modulus) of the spacers and the plate, platethickness, and surface flatness of each plate. Certain findings andpreferred embodiments obtained from the experiments are given below. Thedefinition of spacer height, the IDS, period, and lateral size ofspacers are given in FIG. 16 .

E-1.1 Effects of IDS (Inter-Spacer Distance) and plate thickness andmaterials on sample thickness. Experimentally, we observed that theinter-spacer distance (ISD) of a periodic spacer array can significantlyaffect the sample thickness deviation (from the spacer height) anduniformity that a closed configuration of a CROF process.

FIG. 18 shows the effects of IDS and plate thickness and materials onsample thickness. The measured sample thickness deviation and uniformityvs. inter-spacer distance (IDS) for different plate and spacermaterials, different plate thickness, and different samples. The spacersare a periodic array, and have 5 um spacer height, flat top, and asquare shape (10×10 um pillar lateral size, nearly uniformcross-section, and round corners). The IDS was 20 um, 50 um, 100 um, 200um, and 500 um respectively. The substrate was non-treated 250 um thickPMMA plate of flat surface (1 in×1 in area). The X-Plates, where thespacers were directly fabricated on, was, respectively, non-treated PMMAplate of 175 um and 50 um thick, and non-treated PS of 125 um and 25 umthick. The sample was, respectively, 2 uL blood (dropped by directcontact with finger), saliva, or PBS (dropped by pipette). The CROFdevices were pressed by hand pressing and rubbed over the 1 in by 1 inarea, and were self-hold after the press. The sample thickness weremeasured at the closed configuration of the CROF devices.

FIG. 18 shows that for the given experimental conditions and for thespacer of a square shape (10×10 um pillar lateral size, nearly uniformcross-section, and round corners):

(1) When ISD are 20 um, 50 um, 100 um, the average final samplethickness is 5.1 um to 5.2 um, which is very close to the predeterminedspacer height of 5 um, and has a thickness deviation and uniformity bothless than 4% (namely, if the ISD is equal or less than about 120 um, thedeviation and uniformity can be less than 4%).

(2) But when the ISD is 200 um and 500 um, the average final samplethickness becomes 4.3 um and 3.5 um, respectively, which significantlyless than the predetermined spacer height (5 um), and has a thicknessdeviation of −13.9% and −30.9% and uniformity of 10.9% and 27.7%,respectively. This means that when the ISD is larger than about 200 um,not only the average of the thickness is significantly reduced, but alsothe uniformity becomes very poor.

For a 40 um by 40 um lateral dimension pillar spacer array (FIG. 18 ),when ISD are 60 um and 150 um, 100 um, the average final samplethickness is 5.1 um to 5.2 urn, which is very close to the predeterminedspacer height of 5 um, and has a thickness deviation and uniformity bothless than 4% (namely, if the ISD is equal or less than about 100 um, thedeviation and uniformity can be less than 4%).

E-1.2 Effects of IDS/(Eb{circumflex over ( )}3) on Sample Thickness

Our experiments show (e.g. FIG. 19 ) hat to achieve small samplethickness deviation and good uniformity, the SD⁴/(h^(x)E) (x=1 in theplot) value of X-Plates, should be less than 10{circumflex over ( )}6um{circumflex over ( )}3/GPa, where ISD is inter spacing distance, h isthe height (thickness) of the material, and E is the Young's modulus ofthe material.

In all methods and devices that uses CROF, in certain embodiments,SD4/(hxE) (x=1 in the plot) value is less than 10{circumflex over ( )}6um{circumflex over ( )}3/GPa, less than 5×10{circumflex over ( )}5, lessthan 1×10{circumflex over ( )}6, less than 5×10{circumflex over ( )}6less etc.

In any embodiment, a flexible plates may have a thickness in the rangeof 20 um to 250 um (e.g., in the range of 50 um to 150 um) and Young'smodulus in the range 0.1 to 5 GPa (e.g., in the range of 0.5-2 GPa).

In any embodiment, the thickness of the flexible plate times the Young'smodulus of the flexible plate may be in the range 60 to 750 GPa-urn.

E-1.3 Effects pf Spacer's Size and Height on Sample Thickness

Our experiments show (e.g. FIG. 20 ) that to achieve small samplethickness deviation and for the given plate thickness, the sample, andthe pressing, the IDS should be about 150 um or less.

E-1.4 Effects pf Spacer's Width-to-Height Ratio on Sample Thickness

Our experiments show (e.g. FIG. 21 ) that, to achieve small samplethickness deviation, for the given plate thickness, the sample, and thepressing, and for ISD between 20 um to 150 um, the pillar width toheight ratio (WRH) should larger than 1, and in certain embodiments,preferably equal larger than 2.

This indicates that when the WHR of ˜1 or larger, the spacers are strongenough to sustain the pressing and rubbing in the hand pressing,otherwise the deviation and uniformity will all be poor and large forall of ISD.

E-1.5 Effects pf Spacer's Filling Factors on Sample Thickness

Our experiments show (e.g. FIG. 22 ) that, to achieve small samplethickness deviation and good thickness uniformity, for the given platethickness, the sample, the spacer filling factor should be about 2.3 orlarger.

For example, the less than 4% deviation and uniformity in achieved inFIG. 22 imply that for the given pillar area and IDS, and for the givenspacer area filling factor (i.e. ratio of pillar lateral area to thetotal area), the PS pillars are strong enough to sustain the pressingand rubbing of the hand pressing. The PS pillar deformation can beestimated by the following: the pressure from the thumb is about 1 to 10kg/cm2 (10{circumflex over ( )}5 Pa), the Young's modulus for PS is ˜3GPa, and the filling factor for 20 um width pillar spacer and ISD of 100um is ˜4%, leading the pillar's relative deformation (strain) under thepress of the thumb is 1% to 0.1%, which is consistent to ourexperimental observation.

E-1.6 Effects of Plate Thickness on Sample Thickness

Our experiments show (e.g. FIG. 23 ) that (i) to achieve small samplethickness deviation (equal or less than 5%) and good thicknessuniformity, for the given plate thickness, the sample, at least one ofthe plates should have a plate thickness less than 200 um; and (ii) Ifboth the X-Plate and substrate are thicker than 200 um, they are toorigid, which cannot overcome the dusts, leading to worse spacinguniformity/deviation.

E-1.7. Effects of Substrate Plate on Sample Thickness

Our experiments found (e.g. FIG. 25 ) that if a thicker (1 mm) glasssubstrate plate is used, the maximum IDS for smaller sample thicknessdeviation and good sample thickness uniformity, can extended from 150 umfor PMMA substrate to 200 um.

E-1.8 Plate Surface Wetting Property Modification and Effects onSelf-Holding

Our experiments found (e.g. FIG. 24 ) that: (1) Good self-holding forCROF device requires at least one of the two inner surfaces of the CROFdevice be hydrophilic. (2) If the both inner surfaces of the CROF deviceare hydrophilic, it offers the best self-holding and sample thicknessregulation and uniformity. (3) If one inner surface of the CROF deviceis hydrophilic and the other inner surface is hydrophobic, sample areaneeds to larger than 0.5 cm2 to get a good self-holding. (4) If both ofthe inner surfaces are hydrophobic, the self-hold either poor or fails(unstable). (Lines in figures are for eye-guiding purpose)

E-1.9 Effects of Hand Pressing time on Sample Thickness

Our experiments found that the CROF device can self-holding withpressing time 1 s to 60 s, and have similar good performances. CROFdevice have bad performances and cannot self-hold if no press (press 0s).

E-1.10. Comparison of Effects of Periodic Pillar Spacers to Random BallSpacers on Sample Thickness

The measurements in FIG. 27 show that for the given experimentalconditions, the CROF device with periodic pillar spacer have muchsmaller sample thickness deviation and better uniformity (both less than5%) that the random ball (i.e. beads) spacer. Specifically, for 20 um,50 um and 100 um ISD, the average thickness deviation and uniformitywith periodic, uniform-cross-section pillar spacer are 2.3% and ˜3.4%.However, when using random ball spacer with average ISD of 20 um, 50 umand 100 um, the average thickness deviation and uniformity area 11.2%and 12.5% using the 220 um thick glass cover plate, and 10.8% and 20%using the 175 um thick PMMA cover plate, which are about 5 times largersample thickness deviation and poorer uniformity.

E1.12. Other Findings

FIG. 28 shows the Effects of Different X-Plate Thickness and SubstrateThickness on Sample Thickness.

Our experiments found that the liquid dropped by pipette and direct fromfinger have a similar performance in in the final sample thickness anduniformity.

Our experiments also found that the liquid dropped on Substrate or onX-Plate have similar performances in measured sample thickness anduniformity.

32.2 Complete Blood Count in Undiluted Whole Blood Using Self-HoldingCROF E2.1 CROF Devices Used

The CROF devices, used in all tests in Example 32.2, comprised anX-plate and a flat glass plate. The X-Plate was a 175 um thick PMMA filmof 2.5 cm by 2.5 cm area and having a periodic spacer array in thesample contact area. The glass plate is 1 mm thick and has planarsurface and 3 cm by 5 cm area. The spacers on the X-plate were directlyembossed onto the initially flat PMMA film, hence they were made of PMMA(the same material as the X-plate) and were attached to the X-plate.

Each spacer is a pillar that has nearly uniform lateral cross-section,flat top, and a rectangle shape of 40 um and 30 um width in x and ylateral direction, respectively. All the spacers on a given X-plate havethe same spacer height. The periodic spacer array had a rectanglelattice with a constant period of 120 um and 110 um (in x and y,respectively), giving a constant inter spacer spacing (IDS) of 80 um.

The surfaces of the X-Plate and the glass plate are untreated and arehydrophilic for human blood dropped on the surfaces with a contact angleabout 40 to 50 degrees. Both plates are transparent to visible light.

E2.2 Sample, Preparation, Deposition, CROF Process, Self-Holding

Unless specified otherwise, all blood samples were from healthysubjects, that freshly and directly deposited on a CROF plate, nodilution, and no anti-coagulant added.

In all experiments in Example 3, unless specifically describedotherwise, the blood came from picking human finger, and the blood wasdeposited on a plate of CROF by a direct touching between the blood andthe plate. Typically, the direct touch deposited about 0.1 to 1 uLvolume blood on the surface of the plate. Within about 60 sec after theblood deposition, the CROF process were applied to compressed the bloodsample into a thin film and then perform of the measurements. Unlessspecifically stated, neither anti-conglutination agents nor anyliquid-diluent were added into the blood sample.

In certain experiments where the WBC's need to be stained, prior to ablood testing, the reagent, a dry acridine orange dye layer, wasprecoated on an inner surface (sample contact surface) of one of theplates of the CROF devices. The coating of the dry dye layer comprisedthe steps in sequence: (a) dropping 30 uL acridine orange dye in waterof a 20 ug/mL concentration onto the glass plate, (b) spreading it intoan area of ˜1 cm², and (c) drying for about 1 hour.

Regardless the methods of depositing about 1 uL or less volume blood onone of the CROF plates, the as-deposited blood on the plate formedpuddle of a few millimeter or less diameter. Then the two plates of aCROF device were put into a closed configuration by hand and werepressed by hand for a few seconds, where the original blood puddle wascompressed by the two plates into a large-area thin blood film (about1-3 cm in lateral size). We found that in all of Example 3, unlessdescribed otherwise, the CROF devices pressed by hands had uniformsample thickness regulated by the spacer and were able to self-hold theuniform sample thickness after the hands were released. The samples weredeposited in ordinary room conditions. We found that the dust did notaffect to achieve the predetermined final sample thickness over a largesample area. We also found the that final blood sample spread into arectangle shape with round corners, which we believe, is caused by therectangle lattice of the periodic spacers. This step is illustrated inFIG. 15 .

For the sample using that used the dry dye on the plate, the sample werewaited for 30 sec before any measurements.

The blood sample directly deposited on the plate did not have anydilution by liquid (i.e. no liquid dilution), were only mixed the dryreagent coated the plate. all spacers have the same of rectangle shapeof the same spacer height 2 um height

The 2 um spacer height is selected to make the final blood samplespacing at the closed configuration of CROF device to about 2 um, whichis about equal to the thickness of the red blood cells (RBCs) (2-2.5 μm)but much less than the diameter of RBC (6.2-8.2 μm). Such final samplethickness makes that at the closed configuration of CROF, each RBC iswell separated from others and there is not overlap or rouleaux betweenthe different RBC, allowing accurate accounting of RBC by taking animage of the sample area.

E2.3 Imaging of Blood Cells

The imaging of the blood samples in Example 3 were performed, unlessstated otherwise, with the samples between two CROF plates which were ina closed configuration, and by using commercial DSLR camera (Nikon) andiPhone respectively. The results from each type of cameras are similar.Unless stated otherwise, the images are the top view of the samplesthrough one of the plates which is transparent (i.e. two dimensionalimages in the sample in a plane that is parallel to the surface of theplates).

Nikon Camera. The samples are observed by normal commercial DSLR camera(Nikon) with two filters (a 470±20 nm band pass filter as the excitationfilter and a 500 nm long pass filter as the emission filter), one lightsource (a Xenon lamp) and a magnification/focus lens set. In brightfield mode, a broadband white light source without using any of thefilters. In fluorescence mode, the 470±20 nm filter was placed in frontof the Xenon lamp to create a narrow band excitation source round 470 nmwavelength, and the 500 nm long pass filter was placed in front of thecamera to block the light with wavelength less than 500 nm entering thecamera.Mobile-Phone. iPhone-6 was used for our experiments.

E2.4 Spacer Height (Sample Thickness) Effects on Blood Cells and RBCCounting

In our experiments, the sample thicknesses were controlled to be thesame as the spacer height. We experimentally investigated the effects ofthe spacer height (hence the sample thickness) in a CROF process on theblood cells as well as their imaging and counting. The CROF devices andprocess used and the blood deposition were those described in beginningof this section of Example 2. The blood samples were from the samehealthy subject. Four different spacer heights (1 um, 2 um, 3 um and 5um) were tested in one of our experiment.

FIG. 29 shows the top view optical micrographs (bright field opticalmicroscope) of a blood sample that was CROFed inside four different CROFdevices, where each CROF device has a rectangle lattice of periodicspacers and a different constant spacer height: 1 um (a), 2 um (b), 3 um(c), and 5 um (d). The blood sample were directly deposited by a subjectfinger to a plate of the CROF device, and neither anti-congluent norliquid diluent were added into the blood.

In a bright field optical microscopy, the RBC cells can be seen mucheasier than the WBCs. The red blood cells (RBCs), also termederythrocytes, has a disk diameter of approximately 6.2-8.2 μm and athickness at the thickest point of 2-2.5 pm (near the rim of the disk)and a minimum thickness in the center of 0.8-1 μm.

Our optical microscope observation showed that for 1 um spacer height,˜99% of the RBCS are lysed. For example, FIG. 29(a) showed only RBC leftin the observation field. The 1 um spacer height is significantly lessthan the average RBC thickness. This experiment demonstrated that CROFdevices and process can be used to lyse a cell, by making the finalplate spacer (through a control of the spacer height) less than aminimum dimension of the cell.

Our optical microscope observation (e.g. FIG. 29 ) showed that for the 2um spacer height (sample thickness), the RBCs are all separated fromeach other, have virtually no overlaps between them, and have a shape ofnearly round and symmetrical. The separation between each RBC areclearly seen by a complete-circular dark boundary line of each RBC (i.e.the boundary line completely circulates each (and only one) cell) in the2D microscope image Furthermore, the microscope observation also shownthat the center of the RBCs are darker in the center of cells than thatof the rim, indicating that at 2 um spacer height (sample thickness),the center of the RBCs is still thinner than that at the rim.

Our optical microscope observation showed that (e.g. FIG. 29 ) that whenthe spacer height (hence the sample thickness) was 3 um, the image ofthe blood sample was drastically different from that at 2 um spacerheight in several ways, including, but not limited to: (1) RBCs becamesignificantly overlap, and most of RBC did not have thecomplete-circular dark boundary line that separates each cell as theyexisted in 2 um spacer height, but rather several RBCs shared a singledark bound line which no long be circular shape; and (2) some of RBCsdid not appear in as nearly as round shape as in 2 um spacer height,rather more elliptical shape, and the center dark disk of each RBC,which was clear in 2 um spacer height, become hard to see. As the spacerheight became 5 um, the RBC had more overlaps and more RBCs hadnon-circular shape and nearly invisible dark center (e.g. FIG. 29 ).

It is well known that in a blood without a confinement in space, theRBCs would like to overlap with each other, (e.g. including rouleaux).By confining a blood sample between two planar plate with a spacing of 2um using the spacer height and the plates, the blood thickness is aboutequal to the thickest point of a RBC (which is 2-2.5 μm), thus at agiven location on the plate surface, the confinement forces only one RBCcan exit between the two plates and forces the RBC oriented with itsdisk parallel to the plate surface, leading to well separation betweenRBCs, the complete-circular dark boundary line, and the nearly roundshape, when viewed using a top view optical microscopy.

As the spacer height and hence the sample thickness becomes larger, suchas in 3 um and 5 um spacer height, the sample thickness allows more thanone RBCs between the two plates in a location of the plates, leading RBCoverlaps and disappearance of well-defined boundary of each RBC; andallows a RBC rotating between the two plate and rotating away from thedisk parallel to the plate surface position, leading to un-circularshape in RBC top view image.

For counting RBC number (e.g. used for RBC concentration measurement),our experimentally clearly shown that making a sample thickness to 2 umthick (e.g. using 2 um spacer height) can be easier and more accuratethan that in that the sample thickness of 3 um and 5 um.

By making a spacer height (hence the spacing between two plates and theblood sample thickness) to be about 2 um, which is about equal to thethickness of the red blood cells (RBCs) (2-2.5 μm) but much less thanthe diameter of RBC (6.2-8.2 μm), the blood cell count is much easilyand more accurate than a larger sample thickness.

At a final sample thickness of (2-2.5 μm), or a preferred 1.9 to 2.2 um,makes that at the closed configuration of CROF, each RBC is wellseparated from others and there is not overlap or rouleaux between thedifferent RBC, allowing accurate accounting of RBC by taking an image ofthe sample area.

On the other hand, we observed the CROF device of 1 um spacer height,most of the RBCs are lysed, but not the WBCs or platelets. In ourexperiments, the optical imaging from the top of the CROF (i.e. the CROFplates are nearly parallel to the imaging plane of the imager ofmicroscope or camera) determined (a) the number of cells in an area and(b) the exact lateral size of the area. A lateral size of CROF devicecan be determined by pre-calibration. Or the lateral size of an area ofa CROF device can be determined during an imaging, using the lateralsize of the spacer as a marker. In our experiments, we used both.

In the experiment of Example 2, for the given CROF plates, the spacingbetween the two CROF plates (hence the blood sample thickness) were thesame as the spacer height within 5% or better. Using this samplethickness information, together with the lateral sizes of a given areadetermined from optical imaging, the sample volume associated with thegiven area was determined, which is equal to the sample lateral areatimes the sample thickness. Knowing the sample volume and the number ofcells inside the volume (determined by imaging), we were able todetermine the concentration of cells in that sample volume.

FIG. 29 b shows (b) the ratio of the red blood cell area (measured from2D top view image) to the total lateral area of CROF plate. The maximumat 2 um plate spacing (i.e. sample thickness), because below 2 um someRBC are lysed and higher than 2 um the RBCs are overlapped and rotated,all of them gives smaller RBC area in the 2D image.

One conclusion from the following experiments is that the CROF-deviceoptimized spacing size for blood cell count (RBC and WBC) is 1.9 um-2.2um, or 2 um to 2.2 um, or 2 um to 2.1 um.

Another Our Experimental Finding is that CROF Device with 1 Um SpacingBetween the Two Plates Lyses Most of RBCs, but not Lyses WBCs:CROF-Device

We found that when CROF-device gap spacing is much smaller than thethickness of RBC (e.g. 1 um plate-spacing), the RBC are lysed. WBC ismore elastic, most of them can still be observed, and might be notlysed.

E3.5 Counting RBCs (Red Blood Cells).

In one embodiment, the RBC were counted with bright field mode withoutany filter. A 4×, 10×, 20× or 40× magnification factor were used to takethe pictures. Since both the gap spacing of the X-Plate (t) and thefield of view (A) for each magnification are known (the spacers andtheir periods were used as scale-markers (i.e. rulers), the RBCconcentration in the blood sample are calculated. Fox example, for thecount of N RBC in one field of view, the RBC concentration (C) in bloodis C=N/t/A. This calculation approach is also same for WBC, PLTconcentration measurements.

E2.6. Counting WBC and Platelets

Each white blood cell (WBC), also called leukocyte or leucocyte, has atypical disk diameter of approximately 10-15 μm. A typical platelet(PLT) has a typical size of 1-2 um. Since WBCs and PLT do not havevisible pigment on their own, they are hard to be observable in anordinary microscopy than RBC. To make the WBC and PTL be more visible incounting, in one embodiment, they are stained in acridine orange (AO)dye.

Acridine orange is a stable dye that has a natural affinity for nucleicacids. When binding to DNA, AO intercalates with DNA as a monomer andyields intense green fluorescence under blue excitation (470 nmexcitation, 525 nm green emission for WBC). When binding to RNA andproteins it forms an electrostatic complex in a polymeric form thatyields red fluorescence under blue excitation (470 nm excitation, 685 nmred emission for WBC, PLTs). RBCs have no nucleic acids, thus the cannotbe stained. WBCs have nuclei, both DNA and RNA, thus strongly stained.PLTs have the slight amount of RNA, thus weakly stained. See FIG. 33 .

The WBC were counted under fluorescence mode with a 470±20 nm excitationfilter, and the emission filter is a 500 nm long pass filter, and choose4×, 10×, 20× or 40× magnification factors for taking picture. Usingthese embodiments, the WBC and the PLT were proper counted.

E2.7 Measurements of Different WBCs

WBCs can be classified into five main sub-classes: neutrophils,eosinophils, basophils, lymphocytes, and monocytes; or, sometimes threemain classes: Granulocytes, lymphocytes, and monocytes. Theconcentration of each class in a subject blood may have clinicsignificance, since different infection by virus, bacteria, or fungus,or allergy may change the concentration of certain WBC sub-classconcentration.

The WBC are nucleated, which distinguishes them from the anucleated redblood cells and platelet. Furthermore, different sub-class of WBC hasdifferent ratio of DNA vs. RNA and proteins, they can be differentiatedaccordingly by using a proper dye to stain DNA and RNA separately.

For example, AO dye intercalates with DNA as a monomer and yieldsintense green fluorescence under blue excitation (470 nm excitation, 525nm green emission for WBC). When binding to RNA and proteins it forms anelectrostatic complex in a polymeric form that yields red fluorescenceunder blue excitation (470 nm excitation, 685 nm red emission for WBC,PLTs). Thus different WBC will have different R/G color ratio (greenemission vs. red emission) after stained by AO dye.

AO dye can potentially differentiate 3 kinds of WBC: Granulocytes(including Neutrophil, Eosinophil, Basophil), Lymphocytes, Monocytes.Furthermore, we can directly use camera (or iPphone)'s building-in RGBfilter set to distinguish the green and red emission from G channel andR channel from one photo taken. Thus we have no need to use 2 separatefilter sets (as 525 nm and 685 nm band-pass filter).

As shown in FIG. 34 , there are total 594 WBC counted and plotted. Wecan clearly see that the cells cluster into three distinct regions(shaded areas provided as guides for the eye), corresponding to thethree main white cell subpopulations. The percentage of eachsubpopulations is given in the table, matches well with the normal humanblood value.

E2.8. Hematocrit Measurements

The hematocrit (Ht or HCT), also known as packed cell volume (PCV) orerythrocyte volume fraction (EVF), is the volume percentage (%) of redblood cells in blood. In X-CBC setup, we use 2 um gap spacing, whichwill pack every RBC tightly by substrate and X-Plate. Thus the HCT inthis case is equal to the RBC volume over overall blood volume.

E2.10. Dried AO Dye Staining WBC Speed

WBCs were stained dried AO dye after 30 s, 10 min, 30 min, 90 min. In aCROF process with the dried AO dye on the plate surface, AO dye canstain WBCs fully less than 1 min and will not influence others orover-stain other area after long time. Also, because bound AO fluorescesmore intensely than the unbound dye, no washing step was required.

E2.11. Other Non-Fluorescence Dye to Stain WBC

Non-fluorescence dye to stain WBC can simplify the WBC counting setup.Crystal violet or gentian violet (also known as methyl violet 10B orhexamethyl pararosaniline chloride) is a triarylmethane dye can be usedto stain the nucleus of WBC. Similar to AO dye, we dried 1 mg/m L, 30 uLacridine orange dye in water onto the glass slide with area 1 cm² for 1hour. Then, repeat the X-CBC experiment process. WBC will be stained toviolet color. One drawback of this method is hard to differentiate theWBC subpopulations.

E2.12. No Anti-Coagulant Needed Blood Test by CROP

One advantage of the present invention is that no need to useanti-coagulant reagents to help counting, as being observedexperimentally. In our experiments, X-Plates with 2 um, 3 um and 10 umspacing, and 1 cm×1 cm blood area were tested in a CROF device for ablood sample. In a time duration from 0 min to 80 min, in every 10 min,the pictures at 5 typical points from center to edge of the sample weretaken. All the samples tested are without anti-coagulant reagents. Itwas observed that for the given experimental conditions, there were noconglutination of the blood sample at the closed configuration duringthe observation period. This is because that (1) the CROF with ˜2 umspacing (sample thickness at the closed configuration) separate theblood cells from each other, and (2) the plates of the CROF protect themost of the blood cells from the oxygen.

E2.13. Further Experiments in Blood Cell Count Using CROF and iPhone.

In other experiments, we have tested and validated of a technology and acompact easy-to-use device that allow a person to perform blood cellcounting completely by her/himself in under 20 seconds using asmartphone with less than a drop of blood (<1 uL). All a person needs todo is to let a tiny amount (an arbitrary unknown volume) of blood from apricked finger touch a card, close the card, and take a picture with asmartphone.

One aspect of the present invention is the observation that by preciselyreshaping a blood droplet into a uniform blood layer that is only onered-blood-cell thick (˜2 um) and is confined between two plates, itoffers unprecedented advantages in blood cell counting. The advantagesinclude (i) for fresh undiluted whole blood without adding anyanti-coagulant, the blood cells will be well-separated from each other,not coagulated, and hence easily identifiable by imaging; and (ii) thesample has nearly zero evaporation (in the testing area), keeping theblood cell concentration constant over a long period of time. A secondkey technology we developed is termed “compressed regulated open flow”(CROF), that uses a CROF-Card (a foldable, disposable, stamp-sized (1in-wide, paper-thin) plastic film operated by hand) to perform the bloodreshaping, measure the reshaped blood sample thickness (hence volume),and mix (if needed) pre-coated dry reagents into the blood (and completeall functions in one stroke and in under 5 sec). The last twotechnologies reported here are a small-match-box-sized optical adapterfor smartphone imaging, and software for controlling the smartphone andanalyzing images. The method (“blood-cell-counting using CROF andimaging” or BCI) by a smartphone was validated by comparison with astandard commercial machine, a commercial manual hamocytometer, andmicroscope imaging (in place of smartphone). Over 42 tests using twotypes of blood (stored and fresh from a subject) were run for eachmethod, and red blood cells (RBCs), white blood cells (WBCs), platelets,three WBC differentials, hematocrit (HCT), and mean corpuscular volume(MCV) were measured. The validation shows that the BCI by smartphone hasthe accuracy the same as, or even better, than that a commercial manualhemocytometer (can be further improved), and the same day-to-daystability as commercial instruments. Clearly, the BCI technology hasbroad and significant applications in cell imaging, immunoassays,nucleic-acid assays, personal health monitoring, and other bio-chemicaldetections.

The BCI device comprises three hardware components: a disposablestamp-size plastic CROF-Card (1 in by 1 in area, paper-thin), asmartphone, and a match-box-sized optical adapter (1.5×1.5 in×0.7 in(L×W×H)); and software that controls the smartphone, creates userinterface, and analyzes blood cells. All of them (except smartphone)were designed, and developed by the authors. The optical adapter(“Adapter”), which comprises lenses, mirrors, and filters; and isamounted on the smartphone, makes the smartphone's flash and camerabecome the light source and the imager for the testing, respectively.The optical adapter also has a slot for sliding a CROF-Card in a properposition in the front of the camera (FIG. 30 ). An iPhone-6 was used inour current tests.

In a blood test using the BCI (FIG. 30 ), a person first pricks her/hisfinger, then deposits a small amount (arbitrary unknown volume) of theblood (e.g. less than one drop (<1 uL) directly from the finger onto theCROF-Card by touching the card, closes the card, inserts the card intothe optical adapter, and finally takes a picture of the card using thesmartphone. From the pictures taken, the software does analysis andgives blood cell counts and other parameters.

The total time from depositing the blood onto the CROF-Card to thedisplay of the blood cell count results on the smartphone is ˜12 to 19seconds, where 1-2 s for depositing the blood on the CROF-card, 3-5 sfor closing the card, ˜2-4 s for inserting the card into the Adapter,˜3-5 s for taking images, and 3 s for finishing analysis to show bloodcell count results.

One key innovation of the BCI is the CROF-Card technology developed atus [Ref]. The CROF-Card comprises two pieces of thin plastics, eachabout 1 in.×1 in. in area, a paper thick in thickness, hinged withanother piece at one edge (FIG. 30 ) (note the hinge is not necessarybut convenient). The CROF-Card offers the following key functions inhandling the blood sample: (i) spreading quickly (e.g. 1 sec) the bloodsample from the as-deposited shape (e.g. a puddle of 2 mm diameter and0.4 mm height) into a uniform film of 2 um thick (˜1/200 of the originalthickness) over a significant area (˜500 mm2) and confined by the twoplates of the CROF; (ii) stopping any further reduction of the samplethickness once the 2 um thickness is reached; (iii) keeping the uniform2 um thickness even when the hands are released from the compression(i.e. self-holding, which is due to the capillary forces between theblood and the plates); and (iv) preventing sample evaporation at suchthin thickness (i.e. with the confinement by the two plates, theevaporation occurs only the blood film edge, and the testing area of thesample has a zero evaporation over a very long time). Experimentally,using optical interferences (i.e. Fabry-Perot cavity effect from the twoinner surfaces of the CROF-Card), we found that the CROF-Card byEssenlix can keep the uniform thickness at 2 um with 5% (i.e. 100 nm)uniformity at least over a 20 mm by 20 mm area.

The CROF-Card offers several key and unprecedented advantages for theblood cell counting over the existing methods. The most significant oneis our observation that when a blood drop is reshaped into a uniformblood layer that is only one red-blood-cell thick (˜2 um) and isconfined between two plates, (i) the blood cells in fresh undilutedwhole blood without any anti-coagulant, are well-separated from eachother, have zero coagulation, have much less blood cell motion, and areeasily identifiable by imaging; and (ii) the blood sample has nearlyzero evaporation in the testing area, hence keeping the blood cellconcentration in that area constant over a long period of time.

The second key advantage of the CROF-Card is an “automatic” measurementof the blood sample volume (since the sample thickness is determined). Athird advantage is that it uses least amount of blood sample (sincethere are no fluidic inlet or outlet, or any sample transfer channelsand/or devices). Other advantages are (i) it can mix a dry reagent onthe CROF-Card surface with the sample in a few seconds; (ii) it issimple and fast, and operated by hands, and (iii) it is convenient andlow cost.

Although the method of a blood cells counting by imaging a blood sampleconfined between two plates has over 150 years history and is thefoundation of a commercial manual hemocytometer; to our best knowledge,no one has performed blood cell counting using a plate-confined bloodlayer of a uniform thickness that is just one red-blood cell thick, andno one has examined the behavior of blood cells in a uniform confinedblood sample that is at, or around, one red-blood cell thickness. Inprevious imaging-based approaches, because the confinement spacing ofthe blood sample is larger than a red-cell thickness, the blood samplemust be diluted (often uses anti-coagulant) to avoid the overlaps (hencemiscounting) of the red cells.

Our study has observed intriguing behavior of the blood cells in a wholeblood sample that is confined between two plates and has a uniformsample thickness of just one red-blood cell thick or slightly larger orsmaller than that thickness. The blood cell behavior is drasticallydifferent, depending upon the confinement gap of the CROF-Card (i.e. thesample thickness).

Let us first look at a whole blood that is undiluted, freshly from apricked finger onto the CROF-card, and without adding any anti-coagulant(FIG. 31 .a). For a confinement gap of 2 um, the optical microscopyimage shows that all blood cells (RBCs. WBCs, PLTs) are separated fromeach other in the sample plane (i.e. no overlap), and that each RBC hasa well-defined boundary surrounding each cell with a shadowed center,and each boundary does not cross-over into other RBC's boundary.Furthermore, during the imaging, there were almost no observable cellmovements. One explanation for such behavior is that since 2 umconfinement spacing is slightly smaller than the average thickness of ared cell, each RBC is pinched slightly by the confinement plates,leaving no space for other cells to overlap and unable to move. Clearly,the behavior of the cells with 2 um gap gives an optimum condition tocount the cells by imaging.

However, at 2.2 um gap, some RBCs start to overlap with another RBCs,but there is no observable platelet overlap. A possible reason is thatthere is not enough space for platelets to overlap with PLT. For 2.6 umand 3 um gaps, more RBC's overlap, triple RBCs overlaps become visible,and the platelets overlap with RBCs. These overlaps increase with thegap. Counting blood cells by imaging is possible at the gap of 2.2, 2.6and 3 um, but accuracy is getting poorer with the increasing gap. At 5um and 10 um gap, massive numbers of cells overlap (e.g. coagulated),rouleaux of RBCs are visible, and many RBCs have a narrow ellipticalshape, which is due to the rotation of the RBCs relative to the imagingplane (the large gap makes the rotation possible). Clearly, it isextremely difficult, if not impossible, to accurately count the bloodcells at these gaps.

Now let us look at stored undiluted whole blood with anticoagulant(collected subjects by a commercial service (Bioreclamation Inc.)) Ourstudy showed that (FIG. 31 .b) it has a different response to theCROF-Card confinement gap, compared with fresh undiluted blood withoutanticoagulant. For a 2 um gap, the blood cells in stored blood behavesimilar to those for fresh blood without anti-coagulant. But for largergaps, the stored blood with anti-coagulant has different 2D imagebehavior from that of fresh blood without anticoagulant. With theanticoagulant and at larger than 2 um confinement gap, although the RBCsdo not coagulate together, they can (a) overlap on top of each other and(b) rotate into a narrow elliptical shape in 2D top view imaging, all ofwhich greatly degrade cell counting accuracy.

In the blood cell counting by the smartphone BCI, presented here, theconfinement gap of the CROF-Card (hence the sample thickness) was presetat 2 um with an accuracy better than 5%. The sample volume wasdetermined by the sample thickness preset by the CROF-Card and theimages of a relevant area taken by smartphone. The blood cellconcentrations (RBCs, WBCs, PLTs) were determined by counting the cellsin a relevant area from the image taken by smartphone, and then dividingby the relevant volume. The mean corpuscular volume (MCV) of RBCs wasdetermined by measuring the area of each RBC in a 2D top view image andthe average total volume associated with each RBC, while using thepre-set sample thickness of 2 um. The hematocrit was determined from theproduct of MCV and RBC concentration.

For counting the three WBCs differentials (granulocytes, lymphocytes,monocytes), we stained the blood sample by putting a dried acridineorange (AO) dye layer on one of the CROF-Card surfaces. Since the AOstains the nucleic acids and stain DNA and RNA differently, only WBCsand PLT are stained, and are stained differently according to the amountand ratio of the DNA and RNA in each cell, while RBCs are not stained.The difference in staining gives different fluorescence wavelengths(e.g. 525 nm green emission for stained DNA and 685 nm red emission forstained RNA) and intensity, allowing an identification of each of thethree WBCs differential and PLTs. We found that using the CROF-Card, theWBCs were stained by the precoated AO dye layer in less than 5 sec, dueto the small sample thickness and hence a short dye diffusion time. Thedye staining of the WBC and their fluorescence offer, in addition to thebright-field microscopy, another way to measure the WBCs and was used inthe validation below.

The optical adapter allows an effective field of view of 0.84 mm×0.63 mmfor RBCs, 2.8 mm×2.1 mm for WBC, and 0.2 mm radius in circle for PLT.Currently, the optical adapter needs to move in a slider to for takingthe RBCs and WBCs separately, adding an additional ˜5 secs operationtime. In the next generation, a combined optical adapter without theneed for a slider will be developed. All software for image analysis,user interface, and iPhone control were built by writing our own codesand using certain open source codes. Currently, all blood cell analysispresented here were done by our software in less 2 sec from the image tothe blood counts, except the PLT analysis, which will be less than 5 secin our next generation.

To validate the smartphone BCI, we compared it with the following fourdifferent reference methods (RMs). RM-1 used a high resolutionmicroscope microscope (Nikon Diaphot Inverted Microscope) and DSLRcamera (Nikon D5100) rather than the iPhone and the optical adapter toread the CROF-Card for the same reading area as the current iPhone BCI.RM-2 is the same as RM-1, except that the reading area on the CROF-Cardis extended to 3×3 array with a 8 mm period (total 9 reading areas),equally distributed in 16 mm by 16 mm CROF-Card area. RM-3 uses acommercial manual hemocytometer (purchased from Sigma-Aldrich, Z359629)plus imaging by the same microscope and camera as the RM 1 and 2, but 3mm by 3 mm imaging area. The manual hemocytometer has two chambers, each3 mm by 3 mm in the measurements area and 100 um gap). It requires adilution of blood by 100 times and lysing RBCs for measuring PLTs. RM-4uses a commercial PoC blood cell counting machine (made by one of thelargest blood testing instrument companies); which uses a flowcytometer, and is ˜1 cubic-foot in size and weights ˜20 lb, and costs˜$20,000. The PoC machine requires at least 10 uL volume of blood (over10 drops), blood dilutions, three liquid reagents (lysing, dilution, andcleaning), 5 min operation time and 30 minutes of calibration daily. Thecomparisons allow us to examine each individual function as well ascombined effects of the CROF-Card, imaging by optical adapter andsmartphone, and the imaging by microscope on their performances in bloodcell counting.

In the validation, two types of blood were used: (i) stored blood,purchased from a commercial vendor (Boreclamation.inc), that was mixedwith an anti-coagulant (EDTA); and (ii) fresh blood, which was thefinger-picked blood from two volunteers (During each test, the freshlyfinger-pricked blood was immediately and directly deposited from thefinger to (a) the CROF-Card for the CROF-Card testing and (b) a EDTAcoated tube for the commercial PoC and manual hemocytometer. A total 42samples were tested for each method, over a period of several days.

A total 24 samples were tested in 4 different days (3, 3, 3, and 15samples), and the blood samples were from the same lot for the tests infirst three days, but different lot for the last day. In the fresh bloodsamples, total of 18 samples were tested on 3 different days (6, 6, and6 samples).

The test results showed a number of significant facts. (1) For a givenblood sample, the daily average value of the blood cell counts for thesmartphone BCI (p-BCI) and the all four reference methods are inagreement with each other within its perspective daily CV (coefficientof variation, ratio of the standard deviation to the average).

(2) The comparison of p-BCI with the RM-1 showed that for a givenCROF-Card sample, the blood cell counting using the iPhone and theoptical adapter we developed has the same accuracy (CV) as using thehigh-resolution microscope and DSLR camera (e.g. both have a CV of ˜12%for RBCs) (FIG. 32).

(3) The comparison of RM-1 and RM-2 showed that an imaging of themultiple fields of CROF-Card offers better accuracy than the currentimaging of a single field. CV was improved from ˜12% to ˜6% for RBCs.The multiple field viewing capability will be implemented in our nextgeneration of smartphone BCI for a higher accuracy.

(4) The comparison of RM-2 and RM-3 showed that (i) the CROF-Card is notonly much simpler to use, but also gives a cell counting accuracy thatis the same as or better than the commercial manual hemocytometer inblood cell counting, and (ii) considering the fact in (i) and thecomparison of RM1 and RM2, it lead to the conclusion that a multi-fieldsmartphone BCI should have the same as, or even better, accuracy thanthe commercial manual hemocytometer in blood cell counting. We wouldlike also point out that the variation, although the same for thecurrent CROF-Card and the manual hemocytometer, comes from differentreasons. For the hemocytometer, the variation comes from the dilution,lysing, and manual counting, But for the CROF-Card, the currentvariation (˜7% for RBCs) is mainly due to the sample thickness variation(˜5%), which can be improved further.

(5) The smartphone BCI can identify each of three WBC differentials bystaining and measure the ratio of the intensity of each WBC cell as afunction of the ratio of the fluorescent intensity of green color to redcolor. The standard deviation is similar to that for other blood cellmeasurements. This is because that each sub-type of white blood cellshas a special ratio in fluorescence color (depending their relativeamount and ratio of RNA (red fluorescence) and DNA (green fluorescence);granulocyte has large amounts of RNA and granules (thus high redfluorescence and low green fluorescence); lymphocyte has low amounts ofRNA and high amounts of DNA (thus low red, but high green fluorescence);and monocyte has a red to green ratio between granulocyte andlymphocyte.

(6) Within the statistical significance, the inter-day (i.e. day-to-day)variations for all five methods tested are essentially the same,indicating that the smart-BCI is very stable over the multiple dayperiod that the tests were conducted.

And finally, (7) with our current optical imaging hardware and software,the blood cell counting by imaging is not yet as accurate as thecommercial flow cytometer PoC machine (e.g. ˜7% vs. 1% for RBCs).However, one must recognize two important facts: (i) just with thecurrent accuracy, the p-BCI demonstrated here already has significantvalue in monitoring health and clinical value in the remote area or thedeveloping countries, and (ii) the accuracy of the p-BCI can be furtherimproved to have better accuracy. Undoubtedly, the BCI technology hasbroad and significant applications in cell imaging, immunoassays,nucleic-acid assays, personal health monitoring, and other bio-chemicaldetections.

Certain aspects of the present invention have been described in thefollowing documents and all of these documents are incorporated byreference for all purposes:

U.S. application Ser. No. 13/838,600, filed Mar. 15, 2013 (NSNR-003),which application claims the benefit of U.S. provisional applicationSer. No. 61/622,226 filed on Apr. 10, 2012, and is acontinuation-in-part of U.S. patent application Ser. No. 13/699,270,filed on Jun. 13, 2013, which application is a § 371 filing ofUS2011/037455, filed on May 20, 2011, and claims the benefit of U.S.provisional application Ser. No. 61/347,178, filed on May 21, 2010;

U.S. application Ser. No. 13/699,270, filed Jun. 13, 2013 (NSNR-001),which application is a § 371 filing of international application serialno. US2011/037455, filed on May 20, 2011, which application claims thebenefit of U.S. Provisional Patent Application Ser. No. 61/347,178 filedon May 21, 2010; and

U.S. provisional application Ser. No. 61/801,424, filed Mar. 15, 2013(NSNR-004PRV), provisional application Ser. No. 61/801,096, filed Mar.15, 2013 (NSNR-005PRV), provisional application Ser. No. 61/800,915,filed Mar. 15, 2013 (NSNR-006PRV), provisional application Ser. No.61/793,092, filed Mar. 15, 2013 (NSNR-008PRV), provisional ApplicationSer. No. 61/801,933, filed Mar. 15, 2013 (NSNR-009PRV), provisionalApplication Ser. No. 61/794,317, filed Mar. 15, 2013 (NSNR-010PRV),provisional application Ser. No. 61/802,020, filed Mar. 15, 2013(NSNR-011PRV) and provisional application Ser. No. 61/802,223, filedMar. 15, 2013 (NSNR-012PRV).

Further examples of inventive subject matter according to the presentdisclosure are described in the following enumerated paragraphs.

As used herein, the terms “adapted” and “configured” mean that theelement, component, or other subject matter is designed and/or intendedto perform a given function. Thus, the use of the terms “adapted” and“configured” should not be construed to mean that a given element,component, or other subject matter is simply “capable of” performing agiven function but that the element, component, and/or other subjectmatter is specifically selected, created, implemented, utilized,programmed, and/or designed for the purpose of performing the function.It is also within the scope of the present disclosure that elements,components, and/or other recited subject matter that is recited as beingadapted to perform a particular function may additionally oralternatively be described as being configured to perform that function,and vice versa. Similarly, subject matter that is recited as beingconfigured to perform a particular function may additionally oralternatively be described as being operative to perform that function.

As used herein, the phrase, “for example,” the phrase, “as an example,”and/or simply the terms “example” and “exemplary” when used withreference to one or more components, features, details, structures,embodiments, and/or methods according to the present disclosure, areintended to convey that the described component, feature, detail,structure, embodiment, and/or method is an illustrative, non-exclusiveexample of components, features, details, structures, embodiments,and/or methods according to the present disclosure. Thus, the describedcomponent, feature, detail, structure, embodiment, and/or method is notintended to be limiting, required, or exclusive/exhaustive; and othercomponents, features, details, structures, embodiments, and/or methods,including structurally and/or functionally similar and/or equivalentcomponents, features, details, structures, embodiments, and/or methods,are also within the scope of the present disclosure.

As used herein, the phrases “at least one of” and “one or more of,” inreference to a list of more than one entity, means any one or more ofthe entity in the list of entity, and is not limited to at least one ofeach and every entity specifically listed within the list of entity. Forexample, “at least one of A and B” (or, equivalently, “at least one of Aor B,” or, equivalently, “at least one of A and/or B”) may refer to Aalone, B alone, or the combination of A and B.

As used herein, the term “and/or” placed between a first entity and asecond entity means one of (1) the first entity, (2) the second entity,and (3) the first entity and the second entity. Multiple entity listedwith “and/or” should be construed in the same manner, i.e., “one ormore” of the entity so conjoined. Other entity may optionally be presentother than the entity specifically identified by the “and/or” clause,whether related or unrelated to those entity specifically identified.Thus, as a non-limiting example, a reference to “A and/or B,” when usedin conjunction with open-ended language such as “comprising” may refer,in some embodiments, to A only (optionally including entity other thanB); in certain embodiments, to B only (optionally including entity otherthan A); in yet certain embodiments, to both A and B (optionallyincluding other entity). These entity may refer to elements, actions,structures, steps, operations, values, and the like.

In the event that any patents, patent applications, or other referencesare incorporated by reference herein and (1) define a term in a mannerthat is inconsistent with and/or (2) are otherwise inconsistent with,either the non-incorporated portion of the present disclosure or any ofthe other incorporated references, the non-incorporated portion of thepresent disclosure shall control, and the term or incorporateddisclosure therein shall only control with respect to the reference inwhich the term is defined and/or the incorporated disclosure was presentoriginally.

It is believed that the following aspects and/or claims particularlypoint out certain combinations and subcombinations that are directed toone of the disclosed inventions and are novel and non-obvious.Inventions embodied in other combinations and subcombinations offeatures, functions, elements and/or properties may be claimed throughamendment of the present claims or presentation of new claims in this ora related application. Such amended or new claims, whether they aredirected to a different invention or directed to the same invention,whether different, broader, narrower, or equal in scope to the originalclaims, are also regarded as included within the subject matter of theinventions of the present disclosure.

Aspects:

1. A device for staining and analyzing a tissue, comprising:a first plate, a second plate, and spacers, wherein:

-   -   (i) the first and second plates are movable relative to each        other into different configurations, including an open        configuration and a closed configuration;    -   (ii) one or both plates are flexible;    -   (iii) each of the plates has, on its respective surface, a        contact area for contacting the tissue and/or a staining        solution that stains the tissue; and    -   (iv) one or both of the plates comprise the spacers that are        fixed with a respective plate, wherein the spacers have pillar        shape, a predetermined substantially uniform height of 250 um or        less, and wherein at least one of the spacers is inside the        contact area;

wherein in the open configuration, the two plates are separated apart,the spacing between the plates is not regulated by the spacers, and thetissue is deposited on one or both of the plates; and

wherein in the closed configuration, which is configured after thetissue deposition in the open configuration, at least a part of thetissue and the staining solution is sandwiched between the two platesforming a layer of uniform thickness, wherein the uniform thickness ofthe layer is confined by the contact areas of the two plates and isregulated by the plates and the spacers.

2. A kit for staining a tissue, comprising:

-   -   (i) the device of Aspect 1; and    -   (ii) a staining solution.        3. An apparatus for staining a tissue sample contains or is        suspected to contain an analyte, comprising:    -   (i) the device of Aspect 1, and    -   (ii) an imager that images the tissue sample and the analyte.        4. A system for staining and analyzing a tissue, comprising:    -   (i) the device of Aspect 1; and    -   (ii) an imager that images the tissue.        5. A system for staining and analyzing a tissue sample using a        mobile phone comprising:    -   (i) a sample;    -   (ii) the device of Aspect 1; and    -   (iii) a mobile communication device comprising:        -   a. one or a plurality of cameras for the detecting and/or            imaging the sample;        -   b. electronics, signal processors, hardware and software for            receiving and/or processing the detected signal and/or the            image of the sample and for remote communication; and    -   (iv) a light source from either the mobile communication device        or an external source.        6. A method for analyzing a tissue, comprising:    -   (a) obtaining the device of Aspect 1;    -   (b) having the tissue onto one or both pates of the device;    -   (c) having the plates in a closed configuration by applying an        external force over at least part of the plates; and    -   (d) analyzing the sample in the layer of uniform thickness while        the plates are in the closed configuration.        7. A method for rapidly staining and analyzing a tissue sample        using a mobile communication device, comprising:    -   (a) depositing a tissue sample and a staining liquid on the        device of the system Aspect 5, and placing the two plate into a        closed configuration;    -   (b) obtaining a mobile phone that has hardware and software of        imaging, data processing, and communication;    -   (c) assaying by the tissue sample deposited on the device by the        mobile communication device to generate a result; and    -   (d) communicating the result from the mobile phone to a location        remote from the mobile communication device.        8. A method for staining a tissue sample, comprising:    -   (a) obtaining a tissue sample;    -   (b) obtaining a staining liquid;    -   (c) obtaining a first plate and a second plate, wherein:        -   i. the plates are movable relative to each other into            different configurations;        -   ii. one or both plates are flexible;        -   iii. each of the plates has, on its respective surface, a            sample contact area for contacting a tissue sample or a            staining liquid;        -   iv. the sample contact area in the first plate is smooth and            planner;    -   v. the sample contact area in the second plate comprise spacers        that are fixed on the surface and have a predetermined        substantially uniform height and a predetermined constant        inter-spacer distance that is in the range of 7 um to 200 um;    -   (d) depositing the tissue sample and the staining liquid on the        plates when the plates are configured in an open configuration,        wherein the open configuration is a configuration in which the        two plates are either partially or completely separated apart        and the spacing between the plates is not regulated by the        spacers;    -   (e) after (c), using the two plates to compress at last part of        the tissue sample and at least part of the staining liquid into        a closed configuration;        -   wherein in the closed configuration: at least part of the            sample is between the two plates and a layer of at least            part of staining liquid is between the at least part of the            sample and the second plate, wherein the thickness of the at            least part of staining liquid layer is regulated by the            plates, the sample, and the spacers, and has an average            distance between the sample surface and the second plate            surface is equal or less than 250 um with a small variation.            9. In the device of Aspect 1, wherein the staining solution            is for IHC (immunohistochemistry) staining.            10. In the device of Aspect 1, wherein the staining solution            is for detection of a nucleic acid.            11. In the device of Aspect 1, wherein the tissue contains            or is suspected to contain an analyte that is a protein, a            nucleic acid, DNA, RNA, or a cell.            12. The device of Aspect 1, further comprising a dry            staining reagent coated on one or both plates, wherein the            staining solution is a transfer solution that transfer the            dry staining reagent onto the tissue.            13. The device of Aspect 1, wherein the highly uniform            thickness has a value of 50 um or less.            14. The device of Aspect 1, wherein the uniform thickness            has a value in the range of 0.5 um to 10 um.            15. The device of Aspect 1, wherein the materials of the            plate and the spacers are selected from polystyrene, PMMA,            PC, COC, COP, and another plastic.            16. The device of Aspect 1, wherein the inter-spacer            distance is in the range of 1 um to 200 um.            17. The device of Aspect 1, wherein the inter-spacer            distance is in the range of 200 um to 1000 um.            18. The device of Aspect 1, wherein the spacers regulating            the layer of uniform thickness have a filling factor of at            least 1%, wherein a ratio of the spacer contact area to the            total plate area.            19. The device of Aspect 1, wherein for spacers regulating            the layer of uniform thickness, the Young's modulus of the            spacers times the filling factor of the spacers is equal to            or larger than 20 MPa, wherein the filling factor is a ratio            of the spacer contact area to the total plate area.            20. The device of Aspect 1, wherein:    -   (i) the thickness of the flexible plate times the Young's        modulus of the flexible plate is in the range 60 to 750 GPa-um;    -   (ii) the fourth power of the inter-spacer distance (ISD) divided        by the thickness of the flexible plate (h) and the Young's        modulus (E) of the flexible plate, ISD⁴/(hE), is equal to or        less than 5×10⁶ um³/GPa;    -   (iii) the inter-spacer distance is in the range of 1 um to 200        um;    -   (iv) for each spacer, the ratio of the lateral dimension of the        spacer to its height is at least 1;    -   (v) at least a part of the spacers are arranged periodically;        and    -   (vi) the Young's modulus of the spacers times the filling factor        of the spacers is equal to or larger than 2 MPa, wherein the        filling factor is a ratio of the spacer contact area to the        total plate area.        21. The device of Aspect 1, wherein for a flexible plate, the        fourth power of the inter-spacer distance (ISD) divided by the        thickness of the flexible plate (h) and the Young's modulus (E)        of the flexible plate, ISD⁴/(hE), is equal to or less than 5×10⁶        um³/GPa.        22. The device of Aspect 1, wherein one or both plates comprises        a location marker, either on a surface of or inside the plate,        that provide information of a location of the plate.        23. The device of Aspect 1, wherein one or both plates comprises        a scale marker, either on a surface of or inside the plate, that        provide information of a lateral dimension of a structure of the        sample and/or the plate.        24. The device of Aspect 1, wherein one or both plates comprises        an imaging marker, either on surface of or inside the plate,        that assists an imaging of the sample.        25. The device of Aspect 1, wherein the spacers functions as a        location marker, a scale marker, an imaging marker, or any        combination of thereof.        26. The device of Aspect 1, wherein the spacers are pillars with        a cross-sectional shape selected from round, polygonal,        circular, square, rectangular, oval, elliptical, or any        combination of the same.        27. The device of Aspect 1, wherein the tissue is from a nasal        swab, mucus, earwax, a glandular secretion, tumorous tissue, a        throat swab, skin, biopsy, sputum, pus, microbiota, meconium,        breast milk, throat swab, stool samples, connective tissue,        muscle tissue, nervous tissue, epithelial tissue, and cartilage.        28. The device of Aspect 1, wherein a dye for a stain is        selected from the group consisting of Acid fuchsin, Alcian blue        8 GX, Alizarin red S, Aniline blue WS, Auramine O, Azocarmine B,        Azocarmine G, Azure A, Azure B, Azure C, Basic fuchsine,        Bismarck brown Y, Brilliant cresyl blue, Brilliant green,        Carmine, Chlorazol black E, Congo red, C.I. Cresyl violet,        Crystal violet, Darrow red, Eosin B, Eosin Y, Erythrosin, Ethyl        eosin, Ethyl green, Fast green F C F, Fluorescein        Isothiocyanate, Giemsa Stain, Hematoxylin, Hematoxylin & Eosin,        Indigo carmine, Janus green B, Jenner stain 1899, Light green        SF, Malachite green, Martius yellow, Methyl orange, Methyl        violet 2B, Methylene blue, Methylene blue, Methylene violet,        (Bernthsen), Neutral red, Nigrosin, Nile blue A, Nuclear fast        red, Oil Red, Orange G, Orange II, Orcein, Pararosaniline,        Phloxin B, Protargol S, Pyronine B, Pyronine, Resazurin, Rose        Bengal, Safranine O, Sudan black B, Sudan III, Sudan IV,        Tetrachrome stain (MacNeal), Thionine, Toluidine blue, Weigert,        Wright stain, and any combination thereof.        29. The device of Aspect 1, wherein the staining is by a cell        viability dye selected from the group consisting of: Propidium        Iodide, 7-AAD, Trypan blue, Calcein Violet AM, Calcein AM,        Fixable Viability Dyes, SYTO9 and other nucleic acid dyes,        Resazurin and Formazan (MTT/XTT) and other mitochondrial dyes,        and any combination thereof.        30. The device of Aspect 1, wherein the staining solution        comprises antibodies configured to specifically bind to a        protein analyte in the sample.        31. The device of Aspect 1, wherein the staining solution        comprises oligonucleotide probes configured to specifically bind        to DNA and/or RNA in the sample.        32. The method of Aspect 1, wherein each of the spacers function        as a location marker, a scale marker, an imaging marker, or any        combination of thereof.        33. The device of Aspect 1, wherein the spacers have a pillar        shape and have a substantially flat top surface, wherein, for        each spacer, the ratio of the lateral dimension of the spacer to        its height is at least 2.        34. The device of Aspect 1, wherein the thickness of the        flexible plate times the Young's modulus of the flexible plate        is in the range 60 to 750 GPa-um.        35. The device of Aspect 1, wherein the ratio of the width to        the height of the spacer is equal or larger than 2, and a        product of the filling factor and the Young's modulus of the        spacer is 20 MPa or larger, wherein the filling factor is the        filling factor is the ratio of the spacer contact area to the        total plate area.        36. The device of Aspect 1, wherein the fourth power of the        inter-spacer-distance (IDS) divided by the thickness (h) and the        Young's modulus (E) of the flexible plate (ISD⁴/(hE)) is 5×10⁶        um³/GPa or less.        37. The device of Aspect 1, wherein the spacers are        substantially periodic.        38. The device of Aspect 1, wherein a plate has multiple stain        reagent sites, wherein the ratio of the distance between two        neighbor sites to the sample thickness in the closed        configuration is 5 or larger.        39. The device of Aspect 1, further comprising, on one or both        of the plates, a dry stain reagent or a release time control        material or both.        40. The device of Aspect 1, wherein the spacers regulating the        layer of uniform thickness is periodic and have a filling factor        of at least 2%, wherein the filling factor is the filling factor        is the ratio of the spacer contact area to the total plate area.        41. The device of Aspect 1, wherein one or both plates comprises        a scale maker.        42. The device of Aspect 1, wherein the flexible plate has a        thickness in the range of 10 um to 200 um.        43. The device of Aspect 1, further comprising electrodes on one        or both of the first and second plates, and the electrodes        measure the charge, current, capacitance, impedance, or        resistance of the sample, or any combination of thereof.        44. The device of Aspect 1, further comprising an amplification        surface that is a surface enhancing the fluorescence or        luminescence produced by a detection agent.        45. The device of Aspect 1, wherein the first and second plates        are connected by a hinge.        46. The device of Aspect 1, further comprising, on one or both        of the plates, a dry binding site comprising a capture agent, an        antibody, nucleic acid, a labeled reagent or a cell stain.        47. The device of Aspect 1, wherein the spacing are fixed on a        plate by directly embossing the plate or injection molding of        the plate, and wherein the materials of the plate and the        spacers are selected from polystyrene, PMMA, PC, COC, COP, or        another plastic.

We claim:
 1. A device for staining and/or analyzing a tissue,comprising: a first plate, a second plate, and spacers, wherein: (i) thefirst and second plates are movable relative to each other intodifferent configurations, including an open configuration and a closedconfiguration; (ii) one or both plates are flexible; (iii) each of theplates has, on its respective surface, a contact area for contacting thetissue and/or a staining solution that stains the tissue; and (iv) oneor both of the plates comprise the spacers that are fixed with arespective plate, wherein at least one of the spacers is inside thecontact area; wherein in the open configuration, the two plates areseparated apart, the spacing between the plates is not regulated by thespacers, and the tissue is deposited on one or both of the plates; andwherein in the closed configuration, which is configured after thetissue deposition in the open configuration, at least a part of thetissue and the staining solution is sandwiched between the two platesforming a layer of uniform thickness, wherein the uniform thickness ofthe layer is confined by the contact areas of the two plates and isregulated by the plates and the spacers.
 2. A system or apparatus forstaining and/or analyzing a tissue, comprising: (i) the device of claim1; and (ii) an imager that images the tissue.
 3. A method for stainingand/or analyzing a tissue sample, comprising: (a) obtaining a tissuesample; (b) obtaining a staining liquid; (c) obtaining a first plate anda second plate, wherein: i. the plates are movable relative to eachother into different configurations; ii. one or both plates areflexible; iii. each of the plates has, on its respective surface, asample contact area for contacting a tissue sample or a staining liquid;iv. the sample contact area in the first plate is smooth and planner; v.the sample contact area in the second plate comprise spacers that arefixed on the surface and have a predetermined substantially uniformheight and a predetermined constant inter-spacer distance that is in therange of 7 um to 200 um; (d) depositing the tissue sample and thestaining liquid on the plates when the plates are configured in an openconfiguration, wherein the open configuration is a configuration inwhich the two plates are either partially or completely separated apartand the spacing between the plates is not regulated by the spacers; (e)after (c), using the two plates to compress at last part of the tissuesample and at least part of the staining liquid into a closedconfiguration; wherein in the closed configuration: at least part of thesample is between the two plates and a layer of at least part ofstaining liquid is between the at least part of the sample and thesecond plate, wherein the thickness of the at least part of stainingliquid layer is regulated by the plates, the sample, and the spacers,and has an average distance between the sample surface and the secondplate surface is equal or less than 250 um with a small variation.